What's New in 2007

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April 2006 "Pain Relief Drugs from the Sea"
Desley Blanch interviews Dr. Livett about his research and progress with the development of the cone shell analgesic, ACV1, from Conus victoriae. This interview was broadcast on Radio Australia's Innovations program, April 10, 2006. For a printable transcript click here

For further information see Bruce Livett's Research on cone shell venom peptides for treatment of chronic pain conditions at the Department of Biochemistry and Molecular Biology and the Bio21 Institute for Innovation and Entrepreureship at the University of Melbourne.

  • For a free article on theories about pain, click here: Encyclopedia Britannica.

    Analgesic Component of Venom (ACV1) from Cone Snails :
    see Nature Science Update "Snail toxin could ease chronic pain" by Ingrid Holmes


    An "Internet Interview" with Bruce Livett: conducted in February 2001 about his scientific work with cone shells and conotoxins (and his interaction with other malacologists and shell collectors), is now available as a downloadable Adobe pdf file. This extensive Intervista web "interview" conducted by Eduardo Moreira for Callostoma was subsequently published (in condensed form) in American Conchologist Volume 30, Number 1, 2002, pp. 5 & 14.

    For a one-page description of Cone Shells and their Conotoxins click here

    For a video simulation of cone shell envenomaton click here

    Bruce Livett's more recent publications (1998-2006)

    Site Map of Cone Shells and Conotoxins HomePage

       


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    31 December, 2007

      Wishing you a Happy New Year

      Live Cone Snails from the Seychelles

      David Touitou has posted some amazing photos of live cone shells (some on eggs !!) taken during his November 2007 trip to the Seychelles (family trip). Included are photos of the following Conus gubernator leehmani, Conus canonicus, Conus episcopatus, Conus omria, Conus pennaceus, Conus legatus . You can find these on his homepage http://www.seashell-collector.com/index.htm

    26 December, 2007

      The Conidae of South Africa

      ConchBooks have announced a forthcoming book in progress for release in 2008. The Conidae of South Africa by Manuel Tenorio, part 15 in the series A Conchological Iconography.

      Intrathecal Ziconotide trial for treatment of pain

      Ellis DJ, Dissanayake S, McGuire D, Charapata SG, Staats PS, Wallace MS, Grove GW, Vercruysse, P and the Elan Study 95-002 Group (2008) Continuous intrathecal infusion of Ziconotide for treatment of chronic malignant and nonmalignant pain over 12 months: A prospective, open-label study. Neuromodulation 11: 40-49.
      University of California, San Diego, Medical Center, 9500 Gilman Drive, Suite 0924, La Jolla, CA 92093, USA. Email: mswallace@ucsd.edu

      Abstract: Objectives. This study aims to assess the safety and efficacy of long-term intrathecal (IT) ziconotide infusion.
      Materials and Methods. In this prospective study, 155 patients with severe chronic pain (48 with malignant pain, 107 with nonmalignant pain) who had been responsive to short-term IT ziconotide in a double-blind, placebo-controlled study received long-term, open-label IT ziconotide monotherapy. Efficacy assessments included the mean percentage change on the visual analog scale of pain intensity from baseline in the study of origin; safety was monitored by adverse event (AE) reports, periodic laboratory tests, and vital sign measurements.
      Results. At the last available observation, the visual analog scale of pain intensity scores had decreased by a mean of 36.9% from baseline in the short-term trial (N = 144; 95% CI: 30.1–43.7%; p < 0.0001). The mean IT ziconotide dose remained stable over 12 months in the 31 patients who participated in the study for ? one year. Ziconotide-related AEs were reported in 147 out of 155 patients (94.8%); 39.4% of patients discontinued treatment because of AEs, the majority of which were considered ziconotide related.
      Conclusions. Ziconotide IT monotherapy provided patients with analgesia for 12 months in this open-label study, with an acceptable benefit/risk profile and no evidence of tolerance.

    17 December, 2007

      Internet Hawaiian Shell News (IHSN)

      The IHSN is an Internet Magazine that replaced the printed Hawaiian Shell News (HSN) in January 1997. Both are published by Hawaiian Malacological Society. The new IHSN Portal URL (on 1&1 Web Hosting), on which all issues of IHSN from 1996 to date are accessible is http://s190418054.onlinehome.us/index.html.

    • For issues from 1997 to present
    • For issues from 1960 - Dec 1996. All are large Acrobat files and may take some time to download.

    12 December, 2007

      New postage stamp depicting a cone shell

      During 2007, a new postage stamp depicting Conus clenchi was released by Uruguay. Tom Walker again kindly provided me with the image. It has been added to the Cone Shells on Stamps resource and is available for viewing by clicking here.

    5 December, 2007

      Nonclinical safety of Ziconotide

      Skov MJ,a Beck JC,b de Kater AWc and Shopp GM,d (2007) Nonclinical safety of Ziconotide: An intrathecal analgesic of a new pharmaceutical class. Int. J. Toxicol. 26: 411-421.
      a Elan Pharmaceuticals, Inc., South San Francisco, California, USA; b Roche, Palo Alto, California, USA ; c Pain Therapeutics, Inc., South San Francisco, California, USA; d Shopp Nonclinical Consulting, LLC, Boulder, Colorado, USA

      Abstract: Ziconotide, a potent, selective, reversible blocker of neuronal N-type voltage-sensitive calcium channels, is approved in the United States for the management of severe chronic pain in patients for whom intrathecal therapy is warranted, and who are intolerant or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or intrathecal morphine. In the European Union, ziconotide is indicated for the treatment of severe chronic pain in patients who require intrathecal analgesia. Nonclinical investigations of ziconotide included a comprehensive characterization of its toxicology, incorporating acute and subchronic toxicity studies in rats, dogs, and monkeys; reproductive toxicity assessments in rats and rabbits; and mutagenic, carcinogenic evaluations performed in vivo and in vitro. Additional investigations assessed the potential for cardiotoxicity (rats) and immunogenicity (mice, rats, and guinea pigs), and the presence or absence of intraspinal granuloma formation and local cell proliferation and apoptosis (dogs). The resulting nonclinical toxicology profile was predictive of human adverse events reported in clinical trials and consistent with ziconotide's pharmacological activity. Frequently observed nonclinical behavioral effects included tremoring, shaking, ataxia, and hyperreactivity. Occurrences were generally transient and reversible upon cessation of treatment, and intolerable effects occurred at doses more than 45 times the maximum recommended clinical dose. Ziconotide was not associated with target organ toxicity, teratogenicity, or treatment-related gross or histopathological changes; it displayed no mutagenic or carcinogenic potential and no propensity to induce local cell proliferation or apoptosis. Although guinea pigs developed systemic anaphylaxis, antibodies to ziconotide were not detected in mice, rats, or guinea pigs, indicating low immunogenic potential. No evidence of granuloma formation was observed with intrathecal ziconotide treatment. In summary, the results from these nonclinical safety assessments revealed no significant toxicological risk to humans treated with ziconotide as recommended.
      Keywords: Intrathecal; Safety; Toxicology; Ziconotide

      Solid-phase microwave assisted synthesis of Conotoxin MII

      Galanis AS, Albericio F and Grotli M (2007)Development of new synthetic strategies for synthesis of α-conotoxin-MII. Proc. 10th International Congress on Amino Acids and Proteins (ICAAP), Kallithea, Greece August 20–25, 2007, Amino Acids 33 (3) pp. XIV-XV.
      Department of Chemistry, University of Goteburg, Goteborg, Sweden; Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain

      Abstract: Conotoxins form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup a-conotoxins specifically and selectively bind to subtypes of nicotinic acetylcholine receptors (nAchRs), which are targets for treatment of several neurological disorders. Until now, conotoxins have been mainly prepared by Solid Phase Peptide Synthesis (SPPS) combined with formation of the disulfide bridges after releasing the linear conotoxin precursor from the solid support. We have developed a solid-phase microwave assisted synthesis strategy for the preparation of a-conotoxins.
      α-Conotoxin MII (αCtxMII) was used as a model system. The application of microwave heating to solid-phase peptide synthesis is particularly advantageous as the acceleration of coupling and deprotection reactions should lead to shorter cycle times, higher repetitive yields, and ultimately purer peptides. We present herein a comparison of the results of the synthesis by the classical SPPS and the MW-assisted synthesis. Furthermore, a comparison of several protocols for on-bead or in solution preparation of the two αCtxMII disulfide bridges has been performed. The arsenal of Cys protecting groups commercially available allows us to investigate and optimize several disulfide bond formation methods in order to increase the total yield of the synthesis and the purity of the final product.
      The aim of this work is to develop an improved method able to generate conotoxins in high yield and purity. This will overcome a key barrier currently preventing the efficient synthesis of small focused libraries in order to investigate the structure-activity relationship (SARs) of those peptides. The development of general synthetic strategies for the preparation of conotoxins and analogues are essential to efficiently approach important questions within the area of neurobiology and for the development of novel drugs for treatment of various neurological diseases.

      Structural basis for high specificity binding of the J-superfamily conotoxin pl14a to the Kv1.6 channel

      Mondal S and Ramakumar S (2007) Computational analysis of sequences and structures of conotoxins. Proc. 10th International Congress on Amino Acids and Proteins (ICAAP), Kallithea, Greece August 20–25, 2007, Amino Acids 33 (3) p. X.
      Department of Physics; and Bioinformatics Centre, Indian Institute of Science, Bangalore, India

      Abstract: Conus peptides (conopeptides), the main components of Conus venom, represent a unique arsenal of neuropharmacologically active molecules that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes and neuronal receptors.
      From the point of view of protein sequence and structure analysis, conopeptides can serve as attractive systems for the studies in sequence comparison, pattern extraction, structure-function correlations, protein–protein interactions and evolutionary analysis. Despite their importance and extensive experimental investigations on them, they have been hardly explored through in silico methods. The work carried out in our group is perhaps the first attempt at deploying multi-pronged bioinformatics approaches for studies in the burgeoning field of conopeptides. Our contributions to the in silico analysis include, creation of protein sequence patterns and information highlighting the importance of the patterns as gleaned from the literatures for family classification; profile Hidden Markov Model (HMM) and multi-class support vector machines (MC-SVMs) for conotoxin superfamily classification; in silico characterization of I1 and I2 conotoxin superfamilies; studies of interaction with Kv1 channels of typical members of I2 and J conotoxin superfamilies. The sequence patterns and associated documentation files created by us should be useful in protein family classification and functional annotation. Even though patterns might be useful at the family level, they may not always be adequate at the superfamily level due to hypervariability of mature toxins. In order to overcome this problem, we have demonstrated the application of MCSVMs using pseudo-amino acid composition for the successful in silico classification of the mature conotoxins into their superfamilies. We have analyzed the I- and J-conotoxin-superfamily members in greater detail. The present work provides rationale for the high specificity of J-superfamily conotoxin pl14a to Kv1.6 channel relative to other Kv1 channel subtypes (Kv1.1–Kv1.5).

      Characterizing disulfide-bridged peptides in Conus textile venom Quinton L, Demeure K, Dobson R, Gilles N, Gabelica V, De Pauw E. (2007) New method for characterizing highly disulfide-bridged peptides in complex mixtures: application to toxin identification from crude venoms. J Proteome Res. 6: 3216-3223.
      Laboratoire de Spectrométrie de Masse, Centre d'Analyse des Résidus en Traces, Université de Liège, Liège B-4000, Belgium.

      Abstract: Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.

      Biofutur N° 280: Les venins, nouvelles sources de médicaments?

    • Goyffon M, Ménez A (2007) Appareils venimeux et fonction venimeuse. Biofutur 280: 20-23.
      Abstract: Des espèces venimeuses se rencontrent dans tous les grands groupes du règne animal, constituant parfois des ordres entiers, voire tout un embranchement. Appareils venimeux, glandes à venin, dispositifs d'inoculation, venins eux-mêmes témoignent d'une grande diversité. Plus qu'un avantage sélectif, l'appareil venimeux serait un caractère adaptatif, apte à améliorer la capacitéde survie.

    • Mebs D (2007) Venins et toxines, sources de medicaments. Biofutur 280: 24-26.
      Abstract: Les animaux venimeux produisent dans des glandes spécialisées des venins et des toxines qu'ils utilisent pour leur défense ou la capture des proies. Certaines espèces font appel à une autre stratégie : la séquestration de composés toxiques présents dans l'alimentation ou provenant d'une autre source, et utilisés pour leurs propres objectifs. Ces composés, développés au cours de l'évolution, représentent une source de molécules pharmaceutiques nouvelles.

    • Servent D (2007) Les toxines de serpents. Biofutur 280: 27-30.
      Abstract: Les animaux venimeux produisent dans des glandes spécialisées des venins et des toxines qu'ils utilisent pour leur défense ou la capture des proies. Certaines espèces font appel à une autre stratégie : la séquestration de composés toxiques présents dans l'alimentation ou provenant d'une autre source, et utilisés pour leurs propres objectifs. Ces composés, développés au cours de l'évolution, représentent une source de molécules pharmaceutiques nouvelles.

    • Molgó J, Favreau P and Benoit E (2007) Les venins de cônes : une source de peptides à fort potentiel thérapeutique. Biofutur 280: 31-35.
      Abstract: La grande diversité des venins de cônes, mollusques marins carnivores, représente une source abondante de toxines peptidiques. Ces conotoxines, accessibles par la synthèse chimique, possèdent vis-à-vis des différents soustypes de canaux ioniques et de récepteurs membranaires une spécificité peu commune, qui en fait, dans le domaine des neurosciences, des outils pharmacologiques essentiels et une classe émergente d'agents thérapeutiques.

    • Martin-Eaclaire M-F, Bougis P-E(2007) Les toxines des venins de scorpion. Biofutur 280: 35-39.
      Abstract: La fascination et la répulsion qu'exerce le scorpion sur les humains sont inscrites dans la mythologie et l'astrologie depuis l'Antiquité. Le cinéma ne se prive d'ailleurs pas d'exploiter la crainte viscérale suscitée par cet arachnide nocturne, véritable fossile vivant. Redouté dans certaines régions du monde (Afrique du Nord, Amérique latine), son venin est en revanche très recherché en Asie pour ses propriétés médicinales.

    • Escoubas P (2007) Les venins d'araignées : une mine de nouveaux médicaments ? Biofutur 280: 40-44.
      Abstract: Les araignées, souvent craintes ou mal-aimées, sont l'un des groupes d'animaux venimeux dont les venins sont les plus complexes, et montrent une grande diversité pharmacologique. Leurs toxines constituent une ressource largement inexploitée et les avancées récentes laissent envisager des découvertes importantes pour le développement de nouveaux médicaments, en particulier dans le domaine du traitement de la douleur.

    2 December, 2007

      α4/3 Conotoxins - Review

      Ellison M and Olivera BM (2007). alpha4/3 Conotoxins: phylogenetic distribution, functional properties, and structure-function insights. Chem Rec. 2007 Nov 29;7(6):341-353 [Epub ahead of print]
      Department of Biology, University of Utah, Salt Lake City, Utah, 84112.

      Abstract: This review examines the alpha4/3 conotoxins as an example of molecular diversity in a class of compounds that have evolved in a group of closely related species in a single phylogenetic lineage. The species examined belong to Stephanoconus, a clade of Conus, a genus that contains 500-700 different species of carnivorous marine snails. We examine earlier work that describes the identification and characterization of alpha-ImI, the founding alpha4/3 toxin, and two other alpha4/3 toxins, alpha-ImII and alpha-RgIA. These three toxins all inhibit nicotinic acetylcholine receptors (nAChRs) belonging to a subset of nAChRs that are composed of only alpha subunits; they are, however, diverse in terms of the all-alpha subtype they preferentially antagonize and the receptor site that they bind to. We thus speculate that the alpha4/3 toxins may be a rich source of functionally diverse all-alpha subunit nAChR inhibitors. We review extensive work that has established a detailed model for alpha-ImI binding to one of its preferred nAChR subtypes (the alpha7 nAChR) and, by comparing the alpha-ImI, alpha-ImII and alpha-RgIA sequences demonstrate how structural features of alpha4/3 peptides that account for their diverse functional properties can be identified. This approach is extended to derive models of receptor-toxin binding that may account for the different subtype specificities of alpha4/3 peptides. We also speculate on how rational modification of alpha4/3 toxins may allow engineering of ligands with desired subtype specificities. The chemical diversity produced by the closely related animals in Stephanoconus is thus functionally differentiated, although structurally homologous. (c) 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 7: 341-353; 2007: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20131.

      Ziconotide (ω-conotoxin MVIIA)- A Case Report

      Valia-Vera JC, Villanueva VL, Asensio-Samper JM, Lopez-Alarcon MD and de Andres JA (2007). [Ziconotide: an innovative alternative for intense chronic neuropathic pain.] Rev Neurol. 45:665-669. Spanish.
      Consorcio Hospital General Universitario de Valencia, Valencia, Espana.

      Abstract: INTRODUCTION. Intense chronic pain is a very important health problem, as it has a high prevalence (5-10%), a multifactorial aetiology and its management is very often a very complex affair. Treatment of severe cases sometimes requires interventional approaches, such as continuous intrathecal infusion of opioids. CASE REPORT. We report the case of a 38-year-old female with intense neuropathic pain in the lower back and the lower limbs secondary to three operations on the L5-S1 lumbar segment. After implementing several different pharmacological regimes involving both oral and implanted systems (spinal cord stimulation and subarachnoid infusion pump with different pharmacological combinations) with no clinical improvement, intrathecal infusion with ziconotide was included in the protocol. CONCLUSIONS. Ziconotide is the first specific neuronal blocker that acts on the calcium channel by blocking the N-type voltage-dependent calcium channels. It is a new non-opioid analgesic with approved indication in the treatment of intense chronic pain, in patients who require intrathecal analgesics and are refractory to other analgesic treatments. Therefore, we shall have to consider this drug as a therapeutic alternative in patients do not experience sufficient relief with the pharmacological agents and means currently available to treat them.

    1 December, 2007

      Research on Conopeptides as Analgesics

      Richard Lewis, Paul Alewood, David Adams and MacDonald Christie are Chief Investigators in this NHMRC funded Research Program Dissecting Pain Pathways using Venom Peptides. This is a joint project between the Institute for Molecular Bioscience and The School of Biomedical Sciences at the University of Queensland and the Pain Management Research Institute at the University of Sydney.

      "A major aim of this project is to identify and characterise novel venom peptides from Australia’s venomous creatures, especially the cone snails, which may hold therapeutic potential".

      Download a review of venom peptides and their potential as therapeutics:
      Therapeutic Potential of Venom Peptides

    29 November, 2007

      Animal toxins for pain control - Review

      Cury Y and Picolo G. (2006) Animal toxins as analgesics--an overview. Drug News Perspect. 19: 381-392. Review.
      Laboratory of Pathophysiology, Butantan Institute, São Paulo, Brazil. yarac@attglobal.net

      Abstract: Pain is a multidimensional sensory experience, and multiple mechanisms are involved in the generation of pathophysiological nociceptive pain. Identification of the mechanisms and molecular components responsible for pain generation has not only advanced our understanding of pain and its control, but has also led to the selection of new targets for designing novel analgesic drugs. The high selectivity and specificity of animal toxins have enabled their use as potential therapeutics in the treatment of pain and candidates for the development of new analgesic drugs. This review focuses on the use of animal toxins for pain control and examines the possible analgesic mechanisms of these molecules. Copyright (c) 2006 Prous Science. All rights reserved.

    25 November, 2007

      VISAYA November Vol. 2, No. 2 November 2007

      Tenorio MJ, Poppe GT & Tagaro SP (2007) (n. Sp. and ssp. Of Conus) VISAYA 2: (2) November 2007 [Malacological Journal of Conchology, Inc., Cebu, Philippines] Features images of Conus acutangulus Lamarck, 1810 Philippines ; Conus beatrix n. sp. Northern Territory, Darwin, Australia; Conus pagodus Kiener, 1845 Philippines; and Conus praecellens A. Adams, 1854 Philippines (see Plate 4, p. 90).

      New species of Conus described in VISAYA include Conus frausseni, Conus grohi, Conus terryni (vol 1 no.1); Conus medoci, Conus chiapponorum, Conus vulcanus, Conus claudiae, Conus isabelarum, Conus crioulus, Conus suduirauti (Vol 1 no.2); Conus betulinus rufoluteus (Vol.1 no. 4); Conus guidopoppei (Vol.1 no.5) and Conus beatrix (Vol. 2 no.2). For more information visit: http://www.conchology.be/en/shelltopics/visaya/

      α-Conotoxin MII used to detect α-6 nicotinic receptors in the brain
      Given their restricted localization to striatal DA axons, α*-nAChRs are attracting attention as promising targets for selective pharmacotherapies in dopaminergic disorders including nicotine addiction and Parkinson’s disease. α-CtxMII is a selective antagonist for α3/α6*-nAChRs, but since there is negligible presence of the α3-subunit in mouse striatum, α-CtxMII is selective for α6*-nAChRs. The concentration of α-CtxMII used in this study (30 nM) has no reported detectable effects at non-α6/α3-nAChRs and maximally inhibits the α-CtxMII-sensitive component of nicotine-evoked [3H]-DA release from striatal synaptosomes.

      Exley R, Clements MA, Hartung H, McIntosh JM and Cragg SJ (2007). α6-Containing Nicotinic Acetylcholine Receptors Dominate the Nicotine Control of Dopamine Neurotransmission in Nucleus Accumbens. Neuropsychopharmacology. 2007 Nov 21; [Epub ahead of print]
      Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

      Abstract: Modulation of striatal dopamine (DA) neurotransmission plays a fundamental role in the reinforcing and ultimately addictive effects of nicotine. Nicotine, by desensitizing β2 subunit-containing (β2(*)) nicotinic acetylcholine receptors (nAChRs) on striatal DA axons, significantly enhances how DA is released by reward-related burst activity compared to nonreward-related tonic activity. This action provides a synaptic mechanism for nicotine to facilitate the DA-dependent reinforcement. The subfamily of β2(*)-nAChRs responsible for these potent synaptic effects could offer a molecular target for therapeutic strategies in nicotine addiction. We explored the role of α6β2(*)-nAChRs in the nucleus accumbens (NAc) and caudate-putamen (CPu) by observing action potential-dependent DA release from synapses in real-time using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in mouse striatal slices. The α6-specific antagonist α-conotoxin-MII suppressed DA release evoked by single and low-frequency action potentials and concurrently enhanced release by high-frequency bursts in a manner similar to the β2(*)-selective antagonist dihydro-beta-erythroidine (DHbetaE) in NAc, but less so in CPu. The greater role for α6(*)-nAChRs in NAc was not due to any confounding regional difference in ACh tone since elevated ACh levels (after the acetylcholinesterase inhibitor ambenonium) had similar outcomes in NAc and CPu. Rather, there appear to be underlying differences in nAChR subtype function in NAc and CPu. In summary, we reveal that α6β2(*)-nAChRs dominate the effects of nicotine on DA release in NAc, whereas in CPu their role is minor alongside other β2(*)-nAChRs (eg α4(*)), These data offer new insights to suggest striatal α6(*)-nAChRs as a molecular target for a therapeutic strategy for nicotine addiction.

    20 November, 2007

      Knotted conopeptides !

      PLENTY OF KNOTTIN. In a model of the knottin conotoxin gm9a, the protein backbone is shown as a smooth tube, and three disulfide bridges are shown in a ball-and-stick representation. In all knottins, a disulfide bridge (orange) penetrates a macrocycle (blue) formed by two other disulfides and interconnecting backbone segments. Although conotoxin gm9a's backbone is linear (noncyclic), the close proximity between its N- and C-termini shows how cyclic knottins can easily form by ring closure. MOLMOL [J. Mol. Graphics 14: 51 (1996)] Figure by Laurent Chiche (see 'Tying up Loose Ends' by Stu Borman Science and Technology, April 19, 2004 Volume 82, Number 16 pp. 40-42).

      Gracy J1,2, Le-Nguyen D3, Gelly J-C4, Kaas Q5, Heitz A1,2 and Chiche L1,2 (2007) KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007 Nucleic Acids Res. Open Access 10.1093/nar/gkm939 - published 19 November 2007, .
      1Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale, 34090 Montpellier, 2INSERM, U554, Montpellier, 3CNRS, FRE3009; BIORAD; Complex system modelling and engineering for diagnostic, Montpelllier, 4CNRS, UPR 9080, Université Paris 7, Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, Paris, France and 5Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

      Abstract: The KNOTTIN database provides standardized information on the small disulfide-rich proteins with a knotted topology called knottins or inhibitor cystine knots. Static pages present the essential historical or recent results about knottin discoveries, sequences, structures, syntheses, folding, functions, applications and bibliography. New tools, KNOTER3D and KNOTER1D, are provided to determine or predict if a user query (3D structure or sequence) is a knottin. These tools are now used to automate the database update. All knottin structures and sequences in the database are now standardized according to the knottin nomenclature based on loop lengths between knotted cysteines, and to the knottin numbering scheme. Therefore, the whole KNOTTIN database (sequences and structures) can now be searched using loop lengths, in addition to keyword and sequence (BLAST, HMMER) searches. Renumbered and structurally fitted knottin PDB files are available for download as well as renumbered sequences, sequence alignments and logos. The knottin numbering scheme is used for automatic drawing of standardized two-dimensional Colliers de Perles of any knottin structure or sequence in the database or provided by the user. The KNOTTIN database is available at http://knottin.cbs.cnrs.fr.

      Information about folding of conotoxins is here

      Parallels between snake dendrotoxins and cone snail conkunitzins

      Imperial JS, Silverton N, Olivera BM, Bandyopadhyay PK, Sporning A, Ferber M and Terlau H. (2007) On the origins of fish-hunting in venomous cone snails. Proc. Am. Phil. Soc. 151: 185-200.
      Extract: “Although we have established that the conkunitzins are unrelated to pharmacologically analogous conotoxins targeting K channels in other piscivorous Conus venoms, it was unexpected for them to be so strikingly similar to the K-channel-targeted dendrotoxins from mamba snakes. Snakes and this particular group of cone snails may have independently recruited the widely distributed Kunitz domain motif as a Kchannel-targeted toxin in their venoms, an example of convergence between unrelated venomous animals. The ubiquity of extracellular protease inhibitors containing Kunitz domains could have provided a potential starting point for the evolution of K-channel-targeted ligands in venoms of both the African mambas and the most well-known lineage (Pionoconus) of fish-hunting cone snail species. An alternative and intriguing explanation is that endogenous K-channel modulators exist, which are Kunitz domain proteins, and it is from these that toxins like the conkunitzins may be derived.”

    14 November, 2007

      Predicting d-amino acids in conotoxins

      Buczek O, Jimenez EC, Yoshikami D, Imperial JS, Watkins M, Morrison A and Olivera BM (2007). I(1)-superfamily conotoxins and prediction of single d-amino acid occurrence. Toxicon. 2007 Sep 29; [Epub ahead of print]
      Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

      Abstract: The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.

      PrIIIE, a novel conotoxin from Conus parius

      Lluisma AO, López-Vera E, Bulaj G, Watkins M and Olivera BM (2007) Characterization of a novel ψ-conotoxin from Conus parius, Reeve. Toxicon (2007), doi:10.1016/j.toxicon.2007.07.009
      Marine Science Institute, University of the Philippines, Quezon City, Philippines; Department of Biology and Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT, USA and Instituto de Ciencias del Mar Y Limnología, Universidad Nacional Autónoma de México, México City, México

      Abstract: The M-superfamily of conotoxins currently comprises three major groups of peptides (the μ-, κM-, and ψ-families) that share a key structural characteristic, the six-Cysteine motif CC-C-C-CC, but differ with respect to their molecular targets. The ψ-family consists of M-superfamily conotoxins that are nicotinic acetylcholine receptor (nAChR) antagonists. To date, only two ψ-conotoxins, PIIIE and PIIIF, are known, both of which were isolated from a single Conus species, C. purpurascens. In this paper, we report the discovery and initial characterization of a ψ-conotoxin from another Conus species, C. parius, which we designated as PrIIIE. Its amino acid sequence, inferred from a cloned cDNA, differed significantly from those of PIIIE and PIIIF. Its bioactivity was investigated by using the synthetic form of the peptide in mice and fish bioassays. At 2.5 nmole, the synthetic peptide induced flaccid paralysis in goldfish in ca. 4 min but did not induce any remarkable behavior in mice (after i.c. and i.p. injection of up to 10 nmole of peptide) and did not block action potential in directly-stimulated frog muscle preparations. Electrophysiological experiments carried out to measure inhibition of ion currents through mouse nAChR receptors expressed in oocytes revealed that PrIIIE (IC50 ~ 250 nM) was significantly more potent than PIIIE (IC50 ~ 7000 nM) and that PrIIIE showed higher ihhibition potency against the adult-type than the fetal type nAChR. In similar electrophysiological assays, PrIIIE showed no inhibitory effects against the mouse muscle subtype Na+ channel isoform Nav 1.4. The discovery of this ψ-conotoxin from a Conus species that belongs to the subgenus Phasmoconus, which is distinct from and larger than the clade in which C. purpurascens belongs, suggests that greater structural and functional diversity of ψ-conotoxins remains to be discovered from the members of this subgenus.

      Functional similarity between DSP and conotoxin GVIA

      Kouno T, Mizuguchi M, Tanaka H, Yang P, Mori Y, Shinoda H, Unoki K, Aizawa T, Demura M, Suzuki K, Kawano K. (2007) The Structure of a Novel Insect Peptide Explains Its Ca(2+) Channel Blocking and Antifungal Activities. Biochemistry. 2007 Nov 10; [Epub ahead of print]
      Faculty of Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Faculty of Agriculture, Iwate University, Morioka 020-8550, and Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.

      Abstract: Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.

    8 November, 2007

      "Tying the knot": Cyclic cystine knot scaffolds

      Cemazar M, Gruber CW, Craik DJ (2007). Oxidative Folding of Cyclic Cystine Knot Proteins. Antioxid Redox Signal. Oct 25; [Epub ahead of print]
      Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Australia.

      Abstract: Cyclic cystine knot proteins are small but topologically complex molecules that occur naturally in plants and have a wide range of bioactivities that make them interesting from a pharmaceutical perspective. Their remarkable stability is dependent on the correct formation of a knotted arrangement of disulfide bonds. This review reports on studies that have deciphered the pathways to the "tying of the knot." These studies have involved a range of biophysical techniques and suggest that the major intermediate species presented on these pathways are two disulfide native species, which are not necessarily the precursors of the native protein. Structural elucidations of one analogue and one such intermediate have been reported, and they both show highly native-like conformation and native disulfide bond connectivity. Cyclic cystine knot formation has also been shown to be assisted by protein disulfide isomerase. The points summarized in this review will be important to consider in the design of novel pharmaceutically interesting biomolecules based on the cyclic cystine knot motif, which has shown potential as a molecular scaffold because of its exceptional stability.

      See also : Gruber CW, Cemazar M, Heras B, Martin JL, and Craik DJ. (2006) Protein disulfide isomerase: the structure of oxidative folding. Trends Biochem Sci 31: 455–464.

    6 November, 2007

      CyBase: database of cyclic protein sequences

      Wang CKL,1 Kaas Q,1 Chiche L,2 and Craik DJ,1* (2007) CyBase: a database of cyclic protein sequences and structures, with applications in protein discovery and engineering. Nucleic Acid Research Published Online November 5, 2007
      1Institute for Molecular Bioscience, Australian Research Council Special Research, Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland, 4072, Australia and 2Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale, 34090 Montpellier, 3INSERM, U554, Montpellier, France
      *To whom correspondence should be addressed. Tel: +61 7 3346 2019; Fax: +61 7 3346 2029; Email: d.craik@imb.uq.edu.au

      Abstract: CyBase was originally developed as a database for backbone-cyclized proteins, providing search and display capabilities for sequence, structure and function data. Cyclic proteins are interesting because, compared to conventional proteins, they have increased stability and enhanced binding affinity and therefore can potentially be developed as protein drugs. The new CyBase release features a redesigned interface and internal architecture to improve user-interactivity, collates double the amount of data compared to the initial release, and hosts a novel suite of tools that are useful for the visualization, characterization and engineering of cyclic proteins. These tools comprise sequence/structure 2D representations, a summary of grafting and mutation studies of synthetic analogues, a study of N- to C-terminal distances in known protein structures and a structural modelling tool to predict the best linker length to cyclize a protein. These updates are useful because they have the potential to help accelerate the discovery of naturally occurring cyclic proteins and the engineering of cyclic protein drugs. The new release of CyBase is available at http://research1t.imb.uq.edu.au/cybase

      [Using the cyclization tools incorporated into CyBase, Fig. 2(B) in this article shows a model of a cyclic alpha conotoxin MII using its native linear structure as a template (PDB ID: 1MII) which has been cyclized in silico using a seven-residue poly-alanine linker.
      Click for screen .pdf. See also: Clark,R.J., Fischer,H., Dempster,L., Daly,N.L., Rosengren,K.J.,Nevin,S.T., Meunier,F.A., Adams,D.J. and Craik,D.J. (2005) Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII. Proc. Natl Acad. Sci. USA, 39, 13767–13772].

      Prialt (omega-conotoxin MVIIA) elevated to first-line alternative for intraspinal pain treatment

      Deer, T. et al (2007) Polyanalgesic Consensus Conference 2007: Recommendations for the Management of Pain by Intrathecal (Intraspinal) Drug Delivery: Report of an Interdisciplinary Expert Panel Neuromodulation 10: 300–328.

      Abstract:
      Background. Expert panels of physicians and nonphysicians in the field of intrathecal therapies convened in 2000 and 2003 to make recommendations for the rational use of intrathecal analgesics based on the preclinical and clinical literature known up to those times. An expert panel of physicians convened in 2007 to update previous recommendations and to form guidelines for the rational use of intrathecal opioid and nonopioid agents.
      Methods. A review of preclinical and clinical published relevant studies from 2000 to 2006 was undertaken and disseminated to a convened expert panel of physicians and nonphysicians. Focused discussions were held on the rational use of intrathecal agents and a survey asking questions regarding intrathecal therapies management was given to the panelists.
      Results. The panelists, after review of the literature from 2000 to 2006 and discussion, created an updated algorithm for the rational use of intrathecal opioid and nonopioid agents in patients with nonmalignant and end-of-life pain. Of note is that the panelists felt that ziconotide, based on new and relevant literature and experience, should be updated to a line one intrathecal drug.

      Press Release: CHARLESTON, W.Va., Oct. 31 /PRNewswire/ -- The 2007 Polyanalgesic Consensus Panel (PCP) -- a group of leading national pain management physicians from the United States and abroad -- has updated their intraspinal pain treatment guidelines and recommendations. The findings were released this month in Neuromodulation, a neurology and pain publication for physicians. The panel of pain experts revised the guidelines used to determine treatment via intraspinal infusion for patients suffering from severe chronic pain. The updated algorithm includes Ziconotide (PRIALT Elan Corp.) as a first-line alternative for intraspinal infusion to the opioids morphine and hydromorphone.

      Read report here.
      SOURCE Elan Pharmaceuticals, Inc.
      Related links:
      ELAN
      PRIALT

      [Dr. Baldomero M. Olivera, who's research on cone shells and conotoxins led to the discovery of omega-conotoxin MVIIA and the development of Prialt, was named 2007 Scientist of the Year by the Harvard Foundation at Harvard University, and will receive the degree of doctor of science honoris causa from the University of the Philippines on Jan. 16, 2008. Read news release here ]

    5 November, 2007

      Radulae of Conus from India

      Franklin JB,1,3, Fernando SA,1 Chalke BA,2 and Krishnan KS,2,3* (2007) Radular morphology of Conus (Gastropoda: Caenogastropoda: Conidae) from India. Molluscan Research 27: 111-122.
      1 Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai-608 502, Cuddalore, Tamilnadu, India.
      2 Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai-400 005, India.
      3 National Centre for Biological Sciences, TIFR, Old Bellary Road, Bangalore-560 065, India.
      * Corresponding author E-mail: (K. S. Krishnan): ksk@ncbs.res.in.

      Abstract: Radular morphologies of 22 species of the genus Conus from Indian coastal waters were analyzed by optical and scanning electron microscopy. Although the majority of species in the present study are vermivorous, all three feeding modes known to occur in the genus are represented. Specific radular-tooth structures consistently define feeding modes. Species showing similar feeding modes also show fine differences in radular structures. We propose that these structures will be of value in species identification in cases of ambiguity in other characteristics. Examination of eight discrete radular-tooth components has allowed us to classify the studied species of Conus into three groups. We see much greater inter-specific differences amongst vermivorous than amongst molluscivorous and piscivorous species. We have used these differences to provide a formula for species identification. The radular teeth of Conus araneosus, C. augur, C. bayani, C. biliosus, C. hyaena, C. lentiginosus, C. loroisii, and C. malacanus are illustrated for the first time. In a few cases our study has also enabled the correction of some erroneous descriptions in the literature.

      Disulfide isomerase from Conus amadis

      Gowd KH, Krishnan KS and Balaram P (2007). Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation. Mol Biosyst. 3(8):554-566.
      Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.

      Abstract: Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.

    1 November, 2007

      Mitochondrial genome of Conus textile

      Bandyopadhyay PK, Stevenson BJ, Ownby JP, Cady MT, Watkins M and Olivera BM (2007). The mitochondrial genome of Conus textile, coxI-coxII intergenic sequences and Conoidean evolution. Mol Phylogenet Evol. 2007 Aug 24; [Epub ahead of print]
      Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

      Abstract: The cone snails belong to the superfamily Conoidea, comprising approximately 10,000 venomous marine gastropods. We determined the complete mitochondrial DNA sequence of Conus textile. The gene order is identical in Conus textile, Lophiotoma cerithiformis (another Conoidean gastropod), and the neogastropod Ilyanassa obsoleta, (not in the superfamily Conoidea). However, the intergenic interval between the coxI and coxII genes was much longer in C. textile(165bp) than in any other previously analyzed gastropod. We used the intergenic region to evaluate evolutionary patterns. In most neogastropods and three conidean families the intergenic interval is small (<30 nucleotides). Within Conus, the variation is from 130 to 170bp, and each different clade within Conus, has a narrower size distribution. In Conasprella, a subgenus traditionally assigned to Conus, the intergenic regions vary between 200 and 500bp, suggesting that the species in Conasprella are not congeneric with Conus. The intergenic region was used for phylogenetic analysis of a group of fish-hunting Conus, despite the short length resolution was better than using standard markers. Thus, the coxI-coxII intergenic region can be used both to define evolutionary relationships between species in a clade, and to understand broad evolutionary patterns across the large superfamily Conoidea.

      Terebridae, not Conus ! but venomous nevertheless

      Imperial JS, Kantor Y, Watkins M, Heralde FM III, Stevenson B, Chen P, Hansson K, Stenflo J, Ownby JP, Bouchet P and Olivera BM. Venomous auger snail Hastula (Impages) hectica (Linnaeus, 1758): molecular phylogeny, foregut anatomy and comparative toxinology. J. Exp. Zool. (Mol. Dev. Evol.) 308B:744–756
      Department of Biology, University of Utah, Salt Lake City, Utah.

      Abstract: The >10,000 living venomous marine snail species [superfamily Conoidea (Fleming, 1822)] include cone snails (Conus), the overwhelming focus of research. Hastula hectica (Linnaeus, 1758), a venomous snail in the family Terebridae (Mörch, 1852) was comprehensively investigated. The Terebridae comprise a major monophyletic group within Conoidea. H. hectica has a striking radular tooth to inject venom that looks like a perforated spear; in Conus, the tooth looks like a hypodermic needle. H. hectica venom contains a large complement of small disulfide-rich peptides, but with no apparent overlap with Conus in gene superfamilies expressed. Although Conus peptide toxins are densely post-translationally modified, no post-translationally modified amino acids were found in any Hastula venom peptide. The results suggest that different major lineages of venomous molluscs have strikingly divergent toxinological and venom-delivery strategies.

    28 October, 2007

      Folding of conotoxins: disulfide bridge formation

      Bulaj G and Olivera BM (2007). Folding of conotoxins: Formation of the native disulfide bridges during chemical synthesis and biosynthesis of Conus peptides. Antioxid Redox Signal. 2007 Oct 25; [Epub ahead of print]
      Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah., Department of Biology, University of Utah, Salt Lake City, Utah.

      Abstract: Conopeptides from >700 species of predatory marine Conus snails provide an impressive molecular diversity of cysteine-rich peptides. Most of the estimated 50,000-100,000 distinct conopeptides range in size from 10 to 50 amino acid residues, often with multiple posttranslational modifications. The great majority contain from two to four disulfide bridges. As the biosynthetic and chemical production of this impressive repertoire of disulfide-rich peptides has been investigated, particularly the formation of native disulfide bridges, differences between in vivo and in vitro oxidative folding have become increasingly evident. In this article, we provide an overview of the molecular diversity of conotoxins with an emphasis on the cysteine patterns and disulfide frameworks. The conotoxin folding studies reviewed include regioselective and direct oxidation strategies, recombinant expression, optimization of folding methods, mechanisms of in vitro folding, and preliminary data on the biosynthesis of conotoxins in venom ducts. Despite these studies, how the cone snails efficiently produce properly folded conotoxins remains unanswered. As chemists continue to master oxidative folding techniques, insights gleaned from how conotoxins are folded in vivo will likely lead to the development of the new folding methods, as well as shed some light on fundamental mechanisms relevant to the protein folding problem.

      Conantokins from Conus parius

      Teichert RW, Jimenez EC, Twede V, Watkins M, Hollmann M, Bulaj G, Olivera BM (2007). Novel conantokins from Conus parius venom are specific antagonists of NMDA receptors. J Biol Chem. 2007 Oct 25; [Epub ahead of print]
      Biology, University of Utah, Salt Lake City, UT 84112-0840.

      Abstract: We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2 and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a posttranslationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the third position from the N-terminus, where the Gla residue is replaced by either aspartate or by another posttranslationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different posttranslational modifications. Conantokins-Pr1 and -Pr2 adopt a-helical conformations in the presence of divalent cations (Mg2+ and Ca2+), but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-D-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2 and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.

    25 October, 2007

      Conotoxins that interact with sodium channels

      Ekberg J, Craik DJ and Adams DJ (2007). Conotoxin modulation of voltage-gated sodium channels.Int J Biochem Cell Biol. 2007 Sep 14; [Epub ahead of print]
      School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

      Abstract: The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the muO-conotoxins which inhibit VGSC Na(+) conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the muO-conotoxins, which may develop into potential drug leads for the treatment of pain states.

    21 October, 2007

      4th Int. Peptide Symposium, Cairns, 21-25 October , 2007

      The Scientific Program for the 4th Int. Peptide Symposium held in Cairns, 21-25 October , 2007 includes the following conopeptide-related presentations:

      Abstracts:

    • Andrea Robinson: (Monash)
      Robinson AJ, Garland R, Illesinghe J, Guo C, Ahmed A, Van Lierop BJ and Jackson R.
      Applying homogeneous catalysis to the synthesis of peptides. abs#016
    • Chunguang Wang: (Shanghai, China)
      Han Y, Huang F, Jiang H, Liu H, Wang Q, Wang Y, Chi C, Du W and Wang C.
      Purification and structural characterization of a D-amino acid containing conopeptide, marmophine, from Conus marmoreus. abs#036
    • Grzegorz Bulaj: (Utah, USA)
      Bulaj G, Yoshikami D and Olivera BM.
      Development of Conus-Derived Therapeutics for Pain via Exogenomics and Peptide Engineering. abs#037
    • Raymond Norton: (WEHI, Melbourne)
      Norton RS.
      Toxin Structures: Through the Looking Glass. abs#038 (Conotoxin i-RXIA, Conus radiatus) )
    • Richard Lewis: (IMB and Xenome, Brisbane)
      Lewis R, Palant E, Brust A, Colless B, Schmidt P, Fitzpatrick L, McRitchie I and Drinkwater R.
      Xen2174: a novel conopeptide NET inhibitor with analgesic potential. abs#039
    • Richard Clark: (IMB, Brisbane)
      R. J. Clark RJ, Jensen J, Nevin ST, Callaghan BP, Adams DJ and Craik DJ.
      An orally active conotoxin for the treatment of neuropathic pain. abs#075
    • Aline Dantas de Araujo (IMB, Brisbane)
      Dantas De Araujo A and Alewood PF.
      Directed Folding of Toxins Using Selenocysteine abs#112
    • Markus Muttenthaler: (IMB, Brisbane)
      Directed folding of alpha-Conotoxins using selenocysteine. abs#143
    • Helena Safavi-Hemami: (Biochem. Mol. Biol. & Bio21 Institute, Melbourne)
      Safavi-Hemami H, Williamson NA, Purcell AW and Livett BG.
      Profiling and Functional Studies of Venom Isolated from Conus novaehollandiae. abs#152
    • Christina Schroeder: (IMB, Brisbane)
      Schroeder CI, Ekberg J, Nielsen KJ, Adams DA, Loughnan M, Thomas L, Adams DJ, Alewood PF and Lewis RJ.
      Structure and activity of neuronally selective mu-conotoxins from Conus striatus. abs#153
    • Kalyana Bharati Akondie: (IMB, Brisbane)
      Akondi K, Daly N, Craik D, Alewood P.
      Synthesis and Characterization of a novel alpha-conotoxin LeD2. abs#202
    • Kellie Tuck: (Monash and CSIRO, Clayton)
      Tuck KL, Duggan PJ and Graham JE.
      Synthesis of omega-Conotoxin Mimetics in the Development of Novel Analgesics. abs#263
    • Jonas Jensen: (IMB, Brisbane)
      Jensen J, Clark RJ, Nevin ST, Halai R, Adams DJ and Craik DJ.
      Understanding the structure/activity relationships of alpha-conotoxins. abs#324
    • Lena Sorensen (IMB, Brisbane)
      Sorensen L and Alewood PF.
      Alpha-Conotoxin Ca1.1; Synthesis, Structure and Activity. abs#353
    • Jan-Christoph Westermann: (IMB, Brisbane)
      Westermann J, Kaas Q and Craik DJ.
      ConoServer - a database of peptides from cone snails. abs#363

    18 October, 2007

      ConoServer - an online Database

      David Craik and his research team from the University of Queensland have created a database from over 2000 published sequences of peptides from cone snails. This resource called ConoServer provides a comprehensive overview of conopeptide diversity and their uses as drugs, drug leads and diagnostic tools. Well organized and easily navigable, it can be accessed at http:research1t.imb.uq.edu.au/conoserver/

      T-1 Conotoxin lt5d from Conus litteratus

      Liu J, Wu Q, Pi C, Zhao Y, Zhou M, Wang L, Chen S, Xu A, (2007) Isolation and characterization of a T-superfamily conotoxin from Conus litteratus with targeting tetrodotoxin-sensitive sodium channels. Peptides (2007), doi:10.1016/j.peptides.2007.09.006
      Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, People’s Republic of China.

      Abstract: A T-1-conotoxin, lt5d, was purified and characterized from the venom of vermivorous-hunting cone snails Conus litteratus. The complete amino acid sequence of lt5d (DCCPAKLLCCNP) has been determined by Edman degradation. With two disulfide bonds, the calculated average mass is 1274.57 Da, which is confirmed by MALDI-TOF mass spectrometry (average mass 1274.8778). Under whole-cell patch-clamp mode, lt5d inhibits tetrodotoxin-sensitive sodium currents on adult rat dorsal root ganglion neurons, but has no effects on tetrodotoxin-resistant sodium currents. The inhibition of TTX-sensitive sodium currents by lt5d was found to be concentration-dependent with the IC50 value of 156.16nM. Thus, this is the first T-superfamily conotoxin identified to block TTX-sensitive sodium channels.

    17 October, 2007

      T-1 Conotoxins Pu5.1-Pu5.6 from Conus pulicarius

      Peng C, Wu X, Han Y, Yuan D, Chi C, Wang C. (2007) Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning. Peptides 2007 Sep 4; [Epub ahead of print]
      Institute of Protein Research, College of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, China.

      Abstract: Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.

    11 October, 2007

      Conlysin-Mt a cytolytic peptide from Conus mustelinus

      Biggs JS, Rosenfeld Y, Shai Y and Olivera BM. (2007) Conolysin-Mt : A Conus peptide that disrupts cellular membranes. Biochemistry 2007 Oct 10; [Epub ahead of print]
      Department of Biology, University of Utah, Salt Lake City, Utah 84108, and Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

      Abstract: Conus venoms are estimated to comprise over 100,000 distinct pharmacologically active peptides, the majority probably targeting ion channels. Through the characterization of a cytolytic peptide from the venom of Conus mustelinus, conolysin-Mt, we expand the known conopeptide mechanisms to include association with and destruction of cellular membranes. A new 23AA conopeptide, conolysin-Mt has potent hemolytic activity when tested on human erythrocytes. At a concentration of 0.25 muM, the peptide permeabilized both negatively charged prokaryotic (PE:PG) and zwitterionic eukaryotic (PC:cholesterol) model membranes. The affinity constants (KA) of conolysin-Mt for PE:PG and PC:cholesterol model membranes were 0.9 +/- 0.3 x 107 and 3 +/- 1 x 107 M-1, respectively. In contrast, conolysin-Mt exhibited low antimicrobial activity (MIC > 50 muM) against two Escherichia coli strains, with an MIC for the Gram-positive S. aureus of 25-50 muM. The specificity of conolysin-Mt for native eukaryotic membranes is a novel feature of the peptide compared to other well-characterized cytolytic peptides such as melittin.

      I-Superfamily Conotoxin i-RXIA (r11a)

      Buczek BO, Wei D, Babon JJ, Yang X, Fiedler B, Chen P, Yoshikami D, Olivera BM, Bulaj G, Norton RS.(2007) Structure and Sodium Channel Activity of an Excitatory I(1)-Superfamily Conotoxin.Biochemistry. 46: 9929-9940

      Abstract: Conotoxin i-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I1-superfamily, which contains eight cysteine residues arranged in a -C-C-CC-CC-C-C- pattern. i-RXIA (formerly designated r11a) is one of three characterized I1 peptides in which the third last residue is posttranslationally isomerized to the D configuration. Naturally occurring i-RXIA with D-Phe44 is significantly more active as an excitotoxin than the L-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I1-superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that i-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the D-Phe44 and L-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of i-RXIA as a voltage-gated sodium channel; i-RXIA is an agonist, shifting the voltage dependence of activation of mouse NaV1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in i-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.

    10 October, 2007

      20,000 Ligands under the Sea ! A Review

      Olivera BM and Teichert RW (2007) Diversity of the Neurotoxic Conus Peptides. A Model for Concerted Pharmacological Discovery. Review. Molecular Interventions 7:251-260, (2007)
      Department of Biology, University of Utah, Salt Lake City, UT, 84112

      Abstract: Predatory cone snails (genus Conus) produce a rich array of venoms that collectively contain an estimated 100,000 small, disulfide-rich peptides (i.e., conotoxins, or conopeptides). Over the last few decades, the conopeptides have revealed a remarkable diversity of pharmacological function and utility. An evolutionary rationale for the existence of such a large and pharmacologically diverse set of gene products can be premised on the complexity of intra- and interspecies interactions that define the ecology of Conus snails. Insights into these evolutionary trends, moreover, have been exploited with great neuropharmacological success, so that research into the Conus snails effectively recapitulates a new concerted discovery approach, which we discuss here, for developing unique ligands for both laboratory and therapeutic applications. The Conus peptides thus serve as a model system for reaping the pharmacological potential of biodiverse animal lineages.

      Flourescent alpha6 and beta3 nAChRs

      Drenan MR, Nashmi E, Imoukhuede PI, Just H, McKinney S and Lester HA (2007) Subcellular Trafficking, Pentameric Assembly and Subunit Stoichiometry of Neuronal Nicotinic ACh Receptors Containing Fluorescently-Labeled {alpha}6 and {beta}3 Subunits. Molecular Pharmacology (Accepted 11 October, 2007)
      California Institute of Technology, * Address correspondence to: E-mail: lester@caltech.edu

      Abstract: Neuronal nicotinic ACh receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing {alpha}4, {alpha}6, {beta}2, and {beta}3 subunits are expressed in midbrain dopaminergic neurons and are implicated in the response to smoked nicotine. Here we have studied the cell biological and biophysical properties of receptors containing {alpha}6 and {beta}3 subunits by utilizing fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently-tagged {beta}3 subunits were fully functional compared to receptors with untagged {beta}3 subunits. We find that {beta}3 and {alpha}6-containing receptors are highly expressed in neurons, and co-localize with co-expressed, fluorescent {alpha}4 and {beta}2 subunits in neuronal soma and dendrites. Forster resonance energy transfer (FRET) reveals efficient, specific assembly of {beta}3 and {alpha}6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single {beta}3 subunit is incorporated into nAChRs containing this subunit, whereas multiple subunit stoichiometries exist for {alpha}4 and {alpha}6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Further, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of {alpha}6 and {beta}3-containing receptors using TIRF.

      Key words: Nicotinic cholinergic, Func. analysis receptor/ion channel mutants, Fluorescence techniques, Mutagenesis/Chimeric approaches, Drug tolerance/dependence

    7 October, 2007

      Sowerby's Monograph of the genus Conus

      Guido Poppe has made available as an eBook the Thesaurus Conchyliorum (1848-1847) from the Sowerby family. "This extremely rare book is very important in today’s nomenclature because of the many type figures. Only a few complete copies are still existing today and the antiquarian price for a whole set is extremely high. Today the complete work, all text pages and color plates are available for you to buy and download at moderate prices, probably less than at the time of its publication. You can buy the Thesaurus either complete, by volume or by family/genus."

      Volume 3 of this 5 volume set includes Sowerby's "Monograph of the genus Conus" comprising 104 pages, 24 colour Plates containing 601 illustrations. This publication is available to download separately as a 33.5 Mb file in Adobe .pdb format. (Portrait of Sowerby III)

      To purchase any of these historic and conchologically important volumes visit the CONCHOLOGY, INC web site at http://www.conchology.be Click on eBook, and Register or Log-In to purchase and download.

    3 October, 2007



      The Cone Collector #4

      Antonio Monteiro has teamed with good friend Andre Poremski, who worked on the graphics, to give this essential publication for cone shell enthusiasts a professional touch.The new improved newsletter is currently available both in low resolution PDF format (1.8 Mb) and in high resolution PDF format (48 Mb) at the following sites www.theconecollector.net and www.seashell-collector.com

      Articles include -

      • "Who's Who in Cones: Dieter Rockel
      • "Cone Collecting - A Man's Job ?": Antonio Monteiro
      • "Conus aulicus f. gracianus An Exceptional Specimen": Philippe Quiquandon
      • "Australian Corner (#8 on C. anemone; #9 on C. colmani; #9 on C. minnamurra": Jon F. Singleton
      • "A Family Trip to the Maldive Islands...Searching for Conus pennaceus ganensis": Armando Verdasca and Antonio Monteiro
      • "The Works of Hwass (Continued)" - Plates 316,317,318 from Enclyclopedie Methodique
      • "Caribbean Corner - Bahia's variable Archetypus cones" (with plates of C. bertarollae; C.baiano; C.cargilei): Andre Poremski
      • "The T.C. Mystery (Conus(Rhizoconus) tisii Lan, 1978)": Armando Verdasca, António Monteiro & Mike Filmer
      • "Cones on the Web" - a collection of on-line resources and collection websites
      • "Cone Currents - Exceptionally Large Conus ventricosus": courtesy of Andrea Corso
      • "What am I? - An Elusive Conus from the Philippines": Giancarlo Paganelli
      • "What am I? - Mystery Shell from Remote Trinidad": André Poremski
      • "An Unusually Large Conus granulatus Linnaeus, 1758": Julian Joseph
      • Letters to the Editor - concerning C. stramineus and C.catus from John Tucker

      The creation and maintenance of this Internet site involves certain expenses. To cope with this, Antonio is seeking a voluntary contribution from readers. He suggest something between US$5.00 and US$10.00, per year. Subscriptions can be sent to him at the following address: António Monteiro, Rua Carlos Calisto, 3 - 4 E, 1400-043 Lisboa, Portugal. Ideally, money should be sent in cash or through a postal money order. No cheques, please, since cashing these involves heavy expenses. Should anyone wish to transfer money directly into a Bank account, please get in touch with Antonio at a.j.a.monteiro@netcabo.pt for details.

    30 September, 2007

      Contryphans from Conus amadis, Conus lorisii and Conus striatus

      Thakur SS and Balaram P (2007). Rapid mass spectral identification of contryphans. Detection of characteristic peptide ions by fragmentation of intact disulfide-bonded peptides in crude venom. Rapid Commun Mass Spectrom. 21:3420-3426
      Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

      Abstract: The mass spectrometric cleavage of intact disulfide-bonded peptides in Conus venom has been investigated. Contryphans containing a single disulfide bond are shown to fragment preferentially at X-Pro bonds, giving rise to linearized, unsymmetrical cystine peptides, which subsequently fragment by multiple pathways at the disulfide bridge. Cleavage at the disulfide bond can be initiated by initial loss of the C(alpha)H or C(beta)H proton, resulting in distinct product ions, with the subsequent loss of elemental sulfur, H(2)S or H(2)S(2). Contryphans from Conus amadis, Conus loroisii, and Conus striatus are presented as examples, in which detailed assignment of the product ions resulting from tandem mass spectrometric analysis of the intact disulfide is also accomplished. Characteristic fragments arising from conserved contryphan sequences can be used as diagnostic, permitting rapid identification of this class of peptides in crude venom. The observed fragment ions obtained for contryphans in diverse cone snail species are also compared. Copyright (c) 2007 John Wiley & Sons, Ltd.

    12 September, 2007

      Conotoxin MII recognizes alpha6-containing nicotinic receptors

      Grady SR, Salminen O, Laverty DC, Whiteaker P, McIntosh JM, Collins AC and Marks MJ. (2007) The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum. Biochem Pharmacol. 2007 Jul 27; [Epub ahead of print]
      Institute for Behavioral Genetics, University of Colorado, Boulder, CO, USA.

      Abstract: This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [(125)I]-alpha-bungarotoxin and [(3)H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and alpha-conotoxin MII (alpha-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the alpha6 subunit (alpha4alpha6beta2beta3, alpha6beta2beta3, alpha6beta2) and bind alpha-CtxMII with high affinity. One of these three subtypes (alpha4alpha6beta2beta3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for alpha-CtxMII (alpha4beta2, alpha4alpha5beta2)are somewhat more numerous than the alpha6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.

    9 September, 2007

      What is the pain target for alpha-conotoxins ?

      Nevin ST, Clark RJ, Klimis H, Christie MJ, Craik DJ, Adams DJ. (2007) Are {alpha}9{alpha}10 nicotinic acetylcholine receptors a pain target for {alpha}-conotoxins ? Mol Pharmacol. 2007 Sep 5; [Epub ahead of print]
      University of Queensland.

      Abstract: The synthetic alpha-conotoxin Vc1.1 (ACV1) is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally-modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. Recently, it has been proposed that the primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogues vc1a, [P6O]Vc1.1 and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

      Conus: a source of selective sodium channel blockers for pain treatment

      Cummins TR, Sheets PL and Waxman SG. (2007) The roles of sodium channels in nociception: Implications for mechanisms of pain. Pain 131: 243-257. .
      Department of Pharmacology and Toxicology, Stark Neurosciences Research Institute, Indiana University School of Medicine, 950 West Walnut Street, R2 468, Indianapolis, IN 46202, United States.

      Abstract: Understanding the role of voltage-gated sodium channels in nociception may provide important insights into pain mechanisms. Voltage-gated sodium channels are critically important for electrogenesis and nerve impulse conduction, and a target for important clinically relevant analgesics such as lidocaine. Furthermore, within the last decade studies have shown that certain sodium channel isoforms are predominantly expressed in peripheral sensory neurons associated with pain sensation, and that the expression and functional properties of voltage-gated sodium channels in peripheral sensory neurons can be dynamically regulated following axonal injury or peripheral inflammation. These data suggest that specific voltage-gated sodium channels may play crucial roles in nociception. Experiments with transgenic mice lines have clearly implicated Na(v)1.7, Na(v)1.8 and Na(v)1.9 in inflammatory, and possibly neuropathic, pain. However the most convincing and perhaps most exciting results regarding the role of voltage-gated sodium channels have come out recently from studies on human inherited disorders of nociception. Point mutations in Na(v)1.7 have been identified in patients with two distinct autosomal dominant severe chronic pain syndromes. Electrophysiological experiments indicate that these pain-associated mutations cause small yet significant changes in the gating properties of voltage-gated sodium channels that are likely to contribute substantially to the development of chronic pain. Equally exciting, recent studies indicate that recessive mutations in Na(v)1.7 that eliminate functional current can result in an apparent complete, and possibly specific, indifference to pain in humans, suggesting that isoform specific blockers could be very effective in treating pain. In this review we will examine what is known about the roles of voltage-gated sodium channels in nociception.

      [The uO-conotoxin MrVIB from Conus marmoreus has been shown to be 6- to 10-fold more potent at blocking Nav1.8 currents than neuronal TTX-sensitive currents and when infused intrathecally into mice produced substantial analgesia at doses that showed much less severe motor side-effects than lidocaine (Bulaj et al., 2006; Ekberg et al., 2006). In addition, a small molecule inhibitor of Nav1.8 channels, termed A-803467, appears to be both a potent and highly selective inhibitor of Nav1.8 channels. It was able to block spontaneous and evoked action potentials in rat sensory neurons and showed efficacy in a wide range of animal pain models, including models of acute mechanical pain, inflammatory pain and neuropathic pain (Jarvis et al., 2007). These studies suggest that Nav1.8 selective antagonists may have better therapeutic indices for treating pain than the currently available nonselective antagonists of voltage-gated sodium channels].

      • Bulaj G, Zhang MM, Green BR, Fiedler B, Layer RT, Wei S, et al. (2006) Synthetic muO-conotoxin MrVIB blocks TTX-resistant sodium channel NaV1.8 and has a long-lasting analgesic activity. Biochemistry 45:7404–7414.
      • Ekberg J, Jayamanne A, Vaughan CW, Aslan S, Thomas L, Mould J, et al. (2006) muO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits. Proc Natl Acad Sci USA 103:17030–17035.
      • Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, et al. (2007) A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci USA 104:8520-8525.

      Other subtype-selective sodium channel blockers have been identified in Conus kinoshitai (conotoxin KIIIA, Zhang et al 2007), Conus striatus (conotoxin SIIIA, Green et al 2007), Conus consors (mu-conotoxin CnIIIA & CnIIIB, Zhang et al 2006), Conus catus (mu-conotoxin CIIIA, Zhang et al 2006), and Conus magus (mu-conotoxin MIIIA, Zhang et al 2006) .

      See also: Cummins, TR and Rush AM (2007) Voltage-gated sodium channel blockers for the treatment of neuropathic pain. Expert Rev Neurother. 7:1597-1612.

    6 September, 2007

      Marine sourced compounds for neurology

      Martinez A. (2007) Marine-derived drugs in neurology. Curr Opin Investig Drugs. 8: 525-530. Review.
      NeuroPharma, Avda de la Industria 52, 28760 Madrid, Spain. amartinez@neuropharma.es

      Abstract:The oceans provide a rich source of structurally unique compounds that have demonstrated significant biological activities in a range of indications. In particular, the development of marine compounds is emerging as an important field for neurology. Several marine-derived compounds are currently in clinical trials or have been launched for the treatment of neuropathic pain, schizophrenia and Alzheimer's disease. This review describes the development of several of these compounds, specifically covering the conopeptides, anabaseine and omega-3 fatty acids for the potential treatment of various neurological disorders.

    3 September, 2007

      Protein disulfide isomerase from Conus marmoreus

      Wang ZQ, Han YH, Shao XX, Chi CW, Guo ZY.(2007) Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus. FEBS J. 274: 4778-4787.
      Institute of Protein Research, Tongji University, Shanghai, China.

      Abstract:The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.

    30 August, 2007

      Phase II clinical trial of conotoxin MVIIA (Ziconotide/Prialt) combined with intrathecal morphine

      Wallace MS, Kosek PS, Staats P, Fisher R, Schultz DM and Leong M. (2007) Phase II, open-label, multicenter study of combined intrathecal morphine and Ziconotide: Addition of Ziconotide in patients receiving intrathecal morphine for severe chronic pain. Pain Medicine, OnlineEarly ArticlesPublished article online: 28-Aug-2007
      University of California, San Diego School of Medicine, La Jolla, California. .

      Abstract: OBJECTIVE:. To assess the safety and efficacy of adding intrathecal ziconotide to intrathecal morphine in patients being treated with a stable intrathecal morphine dose. DESIGN: Phase II, multicenter, open-label study with a 5-week titration phase and an extension phase. SETTING: Outpatient clinics. PATIENTS: Patients with suboptimal pain relief receiving stable intrathecal morphine doses (2–20 mg/day). INTERVENTIONS: Intrathecal morphine dosing remained constant during the titration phase. Ziconotide therapy began at 0.60 µg/day and was titrated to a maximum of 7.2 µg/day. During the extension phase, ziconotide and intrathecal morphine dosing were adjusted at the investigator’s discretion. OUTCOME MEASURES: Safety was assessed primarily via adverse event reports. Efficacy was analyzed via percentage change on the visual analog scale of pain intensity and in weekly systemic opioid consumption. RESULTS: Twenty-six patients were enrolled. Treatment-emergent adverse events were generally mild or moderate; the most common (?15% of patients in either study phase) study drug-related (i.e., ziconotide/morphine combination [or ziconotide monotherapy in the extension phase only]) events were confusion, dizziness, abnormal gait, hallucinations, and anxiety. The mean percentage improvement in visual analog scale of pain intensity scores was 14.5% (95% confidence interval: ?9.4% to 38.5%) from baseline to week 5 and varied during the extension phase (range: ?0.4% to 42.8%). Mean percentage change from baseline in systemic opioid consumption was ?14.3% at week 5 and varied considerably during the extension phase. CONCLUSIONS: Ziconotide, combined with stable intrathecal morphine, may reduce pain and decrease systemic opioid use in patients with pain inadequately controlled by intrathecal morphine alone.

    29 August, 2007

      An analgesic peptide mu-conotoxin KIIIA from Conus kinoshitai irreversibly blocks neuronal sodium channels

      Zhang M-M, Green BR, Catlin P, Fiedler B, Azam L, Chadwick A, Terlau H, McArthur JR, French RJ, Gulyas J, Rivier JE, Smith BJ, Norton RS, Olivera BM, Yoshikami D and Bulaj G. (2007) Structure/function characterization of mu -conotoxin kiiia, an analgesic, nearly irreversible blocker of neuronal mammalian sodium channels. J. Biol. Chem. 282: 30699-30706.
      Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84108

      Abstract: Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. u-conopeptides previously characterized as inhibitors of TTX-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ~20% of the TTX-resistant sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and and NaV1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Nav1.2 and only partially for Nav1.6. Other isoforms were blocked reversibly: Nav1.3 (IC50, 8uM) and Nav1.4 (IC50, 80 nM). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4: replacement of Trp8 resulted in reversible block of Nav1.2, whereas replacement of Lys7, Trp8, or Asp11, yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Nav1.2, and that further engineering of mu-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

    26 August, 2007

      Convection-enhanced delivery (CED) of omega-conotoxins MVIIA and GVIA provides prolonged seizure protection in rats.

      Gasior M, White N, Rogawski MA. (2007) Prolonged attenuation of amygdala-kindled seizure measures in rats by convection-enhanced delivery of the N-type calcium channel antagonists {omega}-Conotoxin GVIA and {omega}-Conotoxin MVIIA. J Pharmacol Exp Ther. 2007 Aug 23; [Epub ahead of print]
      Cephalon Inc.

      Abstract: Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here we examined whether CED infusion of the N-type calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA can attenuate kindling measures in fully amygdala kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, omega-conotoxin GVIA (0.005, 0.05, 0.5 nmol), omega-conotoxin MVIIA (0.05, 0.15, 0.5 nmol), proteolytically inactivated omega-conotoxin MVIIA (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of omega-conotoxin GVIA and omega-conotoxin MVIIA over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of omega-conotoxins reached a maximum at 48 h post infusion and then gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated omega-conotoxin MVIIA had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high molecular weight presynaptic N-type calcium channel blockers can produce long lasting inhibition of brain excitability and may provide prolonged seizure protection in focal seizure disorders.

      Selective inhibition of alpha-CtxMII-sensitive nAChRs may counter ethanol reinforcement clues

      Lof E, Olausson P, Debejczy A, Stomberg R, McIntosh JM, Taylor JR, Soderpalm B. (2007)Nicotinic acetylcholine receptors in the ventral tegmental area mediate the dopamine activating and reinforcing properties of ethanol cues. Psychopharmacology (Berl). 2007 Aug 17; [Epub ahead of print]
      Inst. Neuroscience and Physiology, Sect. Psychiatry and Neurochemistry, Sahlgrenska Academy, Gothenburg University and Beroendekliniken, Sahlgrenska University Hospital, Gothenburg, Sweden.

      Abstract: RATIONALE: Cues associated with alcohol can elicit craving, support drug-seeking and precipitate relapse. OBJECTIVES: We investigated the possible involvement of nicotinic acetylcholine receptors (nAChRs) in the ventral tegmental area (VTA) in the conditioned reinforcing properties of ethanol-associated stimuli in the rat. MATERIALS AND METHODS: First, using in vivo microdialysis, we analyzed the effect of VTA perfusion of the nonselective nAChR antagonist mecamylamine (MEC) or the selective alpha4beta2* nAChR antagonist dihydro-beta-erythroidine (DHbetaE) on the nucleus accumbens (nAc) dopaminergic response to the presentation of an ethanol-associated conditioned stimulus (CS). Second, rats were trained to associate a tone + light CS with the presentation of 10% ethanol and were subsequently tested on the acquisition of a new instrumental response with conditioned reinforcement (CR) after local VTA infusion of MEC, DHbetaE, or alpha-Conotoxin MII (alpha-CtxMII, a selective alpha3beta2* and alpha6* nAChR antagonist). RESULTS: The ethanol-associated CS elevated nAc dopamine, an effect that was blocked by VTA perfusion of MEC but not DHbetaE. Systemic administration of MEC or local VTA infusion of MEC or alpha-CtxMII selectively blocked ethanol-associated CR, whereas systemic DHbetaE had no effect. CONCLUSIONS: We hypothesize a novel mechanism by which alcohol-associated cues promote drug-seeking behavior via activation of dopamine-stimulating alpha-CtxMII-sensitive nAChRs in the VTA. Pharmacological manipulations of selective nAChRs may thus be possible treatment strategies to prevent cue-induced relapse.

      Slow folding of Conantokin-T from Conus tulipa

      Du D, Bunagan MR, Gai F. (2007) The effect of charge-charge interactions on the kinetics of {alpha}-helix formation. Biophys J. 93:4076-4082
      University of Pennsylvania.

      Abstract: The formation of monomeric alpha-helix represents one of the simplest scenarios in protein folding; however, our current understanding of the folding dynamics of the alpha-helix motif is mainly based on studies of alanine-rich model peptides. To examine the effect of peptide sequence on the folding kinetics of alpha-helices, we studied the relaxation kinetics of a 21-residue helical peptide, Conantokin-T (Con-T), using time-resolved infrared spectroscopy in conjunction with a laser-induced temperature jump (T-jump) technique. Con-T is a neuroactive peptide found in the venom of the piscivorous cone snail Conus tulipa which contains a large number of charged residues. The T-jump relaxation kinetics of Con-T are distinctly slower than those of previously studied alanine-based peptides, suggesting that the folding time of alpha-helices is sequence dependent. Furthermore, it appears that the slower folding of Con-T can be attributed to the fact that its helical conformation is stabilized by charge-charge interactions or salt-bridges. While this finding contradicts an early molecular dynamics simulation, it also has implications for existing models of protein folding.

    14 August, 2007

      Metabolic Pharmaceuticals discontinues clinical trial program for neuropathic pain drug, ACV1 (conotoxin Vc1.1)

      ASX Announcement. Melbourne 14 August 2007: Metabolic Pharmaceuticals reported having received new data regarding the ability of alpha-conotoxin Vc1.1 (ACV1) to block the human alpha9alpha10 nicotinic acetylcholine receptor, the probable target of ACV1.

      Extract:"Despite the Phase 2A trial of ACV1 in sciatic neuropathic pain having been recently completed, the results of that trial (which are yet to be fully analysed) have been overshadowed by the results of contemporaneous in vitro studies on the ability of ACV1 to block the human alpha9alpha10 nicotinic acetylcholine receptor (nAChR), the probable target of ACV1.
      This second study, which was commenced shortly after the alpha9alpha10 nAChR was identified as the probable molecular target for ACV1 in rats, was undertaken to accurately inform dose selection in future clinical trials. While there is normally similar activity of drug candidates across human and rodent receptors, the study results showed that ACV1 is dramatically less able to block the human alpha9alpha10 nAChR than it is to block the equivalent rat receptors."
      What this means for the ACV1 pain programme: "This lower ability of ACV1 to block the human alpha9alpha10 nAChR means that much larger doses of ACV1 than the dose used in the recently completed Phase 2A trial would be necessary to see effects in humans. Doses at the required level are unlikely to be feasible (impractical to inject and cost of goods prohibitive). The Company has therefore concluded that the ACV1 clinical programme is no longer tenable. As a consequence of that decision, the ongoing Phase 2A trial of ACV1 in diabetic neuropathic pain and post-herpetic neuralgia (shingles related pain) will also be stopped. No further ACV1 trials are foreseen."
      "The Company will focus its research and development on the Oral Peptide Delivery Platform, and will continue to search for new drugs to in-license or acquire."

      J-superfamily conotoxin p114a from Conus planorbis blocks nicotinic and potassium Kv1.6 channels

      Mondal S, Babu RM, Bhavna R, Ramakumar S. (2007) In silico detection of binding mode of J-superfamily conotoxin pl14a with Kv1.6 channel. In Silico Biol. 2007 Mar 26;7:0018 [Epub ahead of print]
      Department of Physics, Indian Institute of Science, India. Email: ramak@physics.iisc.ernet.in.

      Abstract: A novel conotoxin pl14a containing 25 amino acid residues with an amidated C-terminus from vermivorous cone snail, Conus planorbis belongs to J-conotoxin superfamily and this is the first conotoxin, which inhibits both nicotinic acetylcholine receptor subtypes and Kv1.6 channel. We have attempted through bioinformatics approaches to elucidate the extent of specificity of pl14a towards Kv1 channel subtypes (Kv1.1-Kv1.6). Our work provides rationale for the relatively high specificity and binding mode of pl14a to Kv1.6 channel.The pl14a peptide contains two types of structural elements, namely the putative dyad (Lys18 and Tyr19) and basic residue ring constituted of arginine residues. We have carried out in silico docking studies so as to assess the contribution of one or combination of both structural elements of pl14a in blocking of Kv1.6 channel. For this purpose, we have built by homology modelling, the theoretical 3D structure of Kv1.6 channel based on the available crystal structure of mammalian shaker Kv1.2 channel. Docking studies suggest that positively charged residues ring may be involved in the blocking mechanism of Kv1.6 channel. The models suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kappaM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. The newly detected pharmacophore for pl14a interacting with Kv1.6 channel provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively-charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profiles against subtypes of the potassium channels for these conopeptides.

    9 August, 2007

      Conotoxins and sodium channels- REVIEW

      Heinemann SH, Leipold E. (2007) Conotoxins of the O-superfamily affecting voltage-gated sodium channels. Cell Mol Life Sci. 64:1329-1340. Review.
      Center of Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena, Drackendorfer Str 1, Jena, Germany. Stefan.H.Heinemann@uni-jena.de

      Abstract: The venoms of predatory cone snails harbor a rich repertoire of peptide toxins that are valuable research tools, but recently have also proven to be useful drugs. Among the conotoxins with several disulfide bridges, the O-superfamily toxins are characterized by a conserved cysteine knot pattern: C-C-CC-C-C. While omega-conotoxins and kappa-conotoxins block Ca(2+) and K(+) channels, respectively, the closely related delta- and microO-conotoxins affect voltage-gated Na(+) channels (Na(v) channels). delta-conotoxins mainly remove the fast inactivation of Na(v) channels and, thus, functionally resemble long-chain scorpion alpha-toxins. microO-conotoxins are functionally similar to micro-conotoxins, since they inhibit the ion flow through Na(v) channels. Recent results from functional and structural assays have gained insight into the underlying molecular mechanisms. Both types of toxins are voltage-sensor toxins interfering with the voltage-sensor elements of Na(v) channels.

    1 August, 2007

      Conotoxin therapeutics - REVIEW

      Grant MA, Shanmugasundaram K and Rigby AC (2007) Conotoxin therapeutics: a pipeline for success? Expert Opinion on Drug Discovery 2 (4) 453-468.
      Division of Molecular and Vascular Medicine, Department of Medicine, Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA. arigby@bidmc.harvard.edu

      Abstract: The pharmacopoeia of conotoxins from the marine snail Conus has evolved with time, providing a myriad of molecular scaffolds on which critical, molecular pharmacophoric descriptors, responsible for mediating conotoxin receptor–target specificity and selectivity have been grafted. Several reports have defined how these critical determinants contribute to refined, subtype-selective receptor recognition. However, the clinical utility of conotoxins is debatable with a single conotoxin, omega-MVIIA (ziconotide), approved by the US FDA. The authors review the present status of conotoxin-based drug discovery efforts, highlighting ongoing preclinical and clinical studies, while discussing strategies that may be necessary to overcome the barriers inherent to peptide therapeutics. Through the beauty of nature and the art of design it should be possible to expand the Conus pipeline.

      Chemical modification of conotoxins

      Craik DJ and Adams DJ (2007). Chemical Modification of Conotoxins to Improve Stability and Activity. ACS Chem Biol. 2(7):457-468.
      Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. Email: dadams@uq.edu.au.

      Abstract: Conotoxins are small disulfide-rich peptides from the venom of cone snails. Along with other conopeptides, they target a wide range of membrane receptors, ion channels, and transporters, and because of their high potency and selectivity for defined subtypes of these receptors, they have attracted a great deal of attention recently as leads in drug development. However, like most peptides, conopeptides potentially suffer from the disadvantages of poor absorption, poor stability, or short biological half-lives. Recently, various chemical approaches, including residue substitutions, backbone cyclization, and disulfide-bridge modification, have been reported to increase the stability of conopeptides. These manufactured interventions add to the array of post-translational modifications that occur naturally in conopeptides. They enhance the versatility of these peptides as tools in neuroscience and as drug leads.

      alpha-conotoxin TxIA from Conus textile selectively targets the alpha3beta2 nAChR subtype.

      Dutertre S, Ulens C, Buttner R, Fish A, van Elk R, Kendel Y, Hopping G, Alewood PF, Schroeder C, Nicke A, Smit AB, Sixma TK and Lewis RJ (2007) AChBP-targeted alpha-conotoxin correlates distinct binding orientations with nAChR subtype selectivity. EMBO J. 26(16):3858-67
      Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.

      Abstract: Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel alpha-conotoxin (alpha-TxIA) in the venom of Conus textile. alpha-TxIA bound with high affinity to AChBPs from different species and selectively targeted the alpha(3)beta(2) nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20 degrees backbone tilt compared to other AChBP-conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.

      Biodistribution of alpha-conotoxin MII after oral administration

      Blanchfield JT, Gallagher OP, Cros C, Lewis RJ, Alewood PF and Toth I (2007). Oral absorption and in vivo biodistribution of alpha-conotoxin MII and a lipidic analogue. Biochem Biophys Res Commun. 361: 97-102.
      School of Molecular and Microbial Sciences, The University of Queensland, Brisbane 4072, Australia.

      Abstract: Conotoxins are highly constrained peptide toxins that exhibit pharmaceutically relevant biological activities. We herein report the extent of absorption and profile of distribution of a native alpha-conotoxin, MII and a lipophilic analogue of MII (N-LaaMII) after intravenous (iv) and oral administration to male Sprague-Dawley rats. N-LaaMII is formed by coupling 2-amino-d,l-dodecanoic acid (Laa) to the N-terminus of MII and has previously been shown to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII while maintaining the potency in inhibition of nAChRs of the parent peptide. Both peptides crossed the GI tract after oral administration ( approximately 6% after 30m). While Laa conjugation did not significantly improve absorption, it did greatly increase the accumulation of the compound in the liver after iv administration. Neither peptide crossed the blood-brain barrier to any significant extent. This is the first study of the in vivo biodistribution of an alpha-conotoxin after oral administration.

      Activities of alpha conotoxins SrIA and SrIB, from Conus spurius

      Lopez-Vera E, Aguilar MB, Schiavon E, Marinzi C, Ortiz E, Restano Cassulini R, Batista CV, Possani LD, Heimer de la Cotera EP, Peri F, Becerril B and Wanke E (2007). Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors. FEBS J. 274(15):3972-3985.
      Laboratorio de Neurofarmacolog­a Marina, Departamento de Neurobiolog­a Celular y Molecular, Instituto de Neurobiolog­a, Universidad‚ Nacional Autnoma de Mexico, Campus Juriquilla, Queretaro, Mexico.

      Abstract: alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.

    29 July, 2007

      Conus peptides targeting pain via alpha9alpha10 nicotinic receptors

      Vincler M, McIntosh JM. (2007) Targeting the alpha9alpha10 nicotinic acetylcholine receptor to treat severe pain. Expert Opin Ther Targets. 11: 891-897.
      Wake Forest University Health Sciences, Department of Anesthesiology, Medical Center Blvd., Winston-Salem, NC 27157, USA.

      Abstract: The alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) are recognized for their function in the hair cells of the inner ear; transcripts for a9 and/or a10 subunits have also been identified in a diverse range of other tissues , including immune cells. The functioning of alpha9alpha10 nAChRs in these latter tissues is unknown. However, a recent series of studies has provided evidence that blockade of the alpha9alpha10 nAChR can alleviate chronic pain resulting from overt peripheral nerve injury or inflammation and increase the functional recovery of damaged neurons. Systemic administration of alpha9alpha10 antagonists produces an acute analgesia; repeated daily administrations produces sustained and cumulative levels of analgesia across 7 days without the development of tolerance. Although the exact mechanism of action is unknown, antagonism of the alpha9alpha10 nAChRs reduces the number of immune cells present at the site of injury.
      Keywords: ACV1, analgesia, conotoxin, neuropathic pain, RgIA, Vc1.1

      Feeding by molluscivorous Conus

      Kantor, Y. (2007) How much can Conus swallow? Observations on molluscivorous species. J. Molluscan Studies Advance Access published online on April 26, 2007
      A.N. Severtzov Institute of Ecology and Evolution, Russian Academy of Sciences, 33 Leninski Prospect 33, Moscow 119071, Russia.
      E-mail: kantor@malaco-sevin.msk.ru

      Abstract: Feeding of three species of molluscivorous Conus, C. textile, C. bandanus and C. omaria, was studied in aquaria. Conus spp. are able to kill and remove from the shell prey larger than themselves. Also, Conus swallowed prey with weight up to half that of the predator. Estimates suggest that molluscivorous species of Conus are probably able to swallow prey with a shell volume reaching 85% of that of the predator, depending on the shape of the prey's body. It is confirmed that the thinning of the inner shell walls in Conus is connected with the ability to swallow voluminous prey. Digestion of prey occurs in both the oesophagus and stomach.

      (See also)

      Feeding by Conus victoriae

      Kohn, AJ (2003) The feeding process in Conus victoriae. In: Wells, FE, Walker, DI and Jones, DS (eds) The Marine Flora and Fauna of Dampier, Western Australia, pp. 101-107. Western Australian Museum, Perth. [Download .pdf file]
      Department of Zoology, University of Washington, Seattle, Washington 98195-1800, U.S.A. Email: kohn@u.washington.edu

      Abstract: The toxoglossan gastropod Conus victoriae preys on other prosobranch gastropods, and its mode of prey capture is shown to be behaviorally more complex and time-consuming than in congeners that feed on polychaetes and fishes. The major difference is that while the latter species capture prey by injecting venom with a single radular tooth, C. victoriae shot five teeth into a single prey snail, Cantharus erythrostomus, at intervals of 5–15 min in the feeding episode observed in an aquarium. The feeding process, over nearly one hour, was videotaped in its entirety from the first evidence of interest in the prey to its final engulfment by the rhynchodaeum. The use of repeated venom injections in capturing a single prey confirms a prediction based on recovery of multiple radular teeth from alimentary tracts and feces of other molluscivorous Conus species, and previous briefly reported observations.

      Recent Updates to The Conus Biodiversity Website

      Dr. Alan Kohn has announced that the Conus Biodiversity Website (http://biology.burke.washington.edu/conus/) now contains “Conus Species Accounts” of the 316 species of Indo-Pacific Conus from the Manual of the Living Conidae, by Röckel, Korn and Kohn (1995). All of the information is databased and tabs above each species facilitate access to the synonymies and subspecies, paleontological records, gene sequences, and the original description. Additions to the site include :

      • 15 videos of Conus feeding biology that show the envenomation and swallowing of prey by representatives of all three major feeding types, those that specialize on polychaetes, on other gastropods, and on fishes. In those cases where the videos have been the subject of published studies, the citations are provided, as are links to available PDFs, and two are shown as links to the Biological Bulletin website.
      • an increased number of images of species-group taxa (800). Most are photographic images of types, while others are representations of holotypes and lectotypes, or figures that accompanied original descriptions but have not been designated as representations of types.
      • 264 PDF’s of works containing the original descriptions of species-group taxa in Conus described between 1758 and 1930, as well as 21 works published since. These may be downloaded by clicking on the color PDF symbol wherever it appears on the web site.
      • 320 PDF’s of additional works containing original descriptions between 1930 and the present. These are not available for electronic download because of copyright protections but a black and white PDF symbol indicates holdings. Help gathering works with original descriptions or filling in missing portions (pages, plates etc.) of PDF’s would be greatly appreciated.

      The Conus Biodiversity Website is based on research supported by the National Science Foundation. Currently this project emphasizes revisionary systematics of Conus in the Western Atlantic region. Additional preserved specimens of Conus, for anatomical and molecular genetic studies are needed. Recently collected specimens preserved in alcohol are most suitable for the latter, but age and mode of preservation is less critical for anatomical study. Dr Kohn would be most grateful to learn of any specimens that might be available for study, either from past collections or that might be collected in the future.

    6 July, 2007

      alphaC-Conotoxin PrXA from Conus parius resembles snake toxin Waglerins

      Jimenez EC, Olivera BM, Teichert RW. (2007) alphaC-Conotoxin PrXA: A New Family of Nicotinic Acetylcholine Receptor Antagonists. Biochemistry. 46: 8717-8724.
      Department of Biology, University of Utah, Salt Lake City, Utah 84112, and Department of Physical Sciences, University of the Philippines Baguio, Baguio City, Philippines.

      Abstract: We have purified a novel paralytic peptide with 32 AA and a single disulfide bond from the venom of Conus parius, a fish-hunting species. The peptide has the following sequence: TYGIYDAKPOFSCAGLRGGCVLPONLROKFKE-NH2, where O is 4-trans-hydroxyproline. The peptide, designated alphaC-conotoxin PrXA (alphaC-PrXA), is the defining member of a new, structurally distinct family of Conus peptides. The peptide is a competitive nAChR antagonist; all previously characterized conotoxins that competitively antagonize nAChRs are structurally and genetically unrelated. (Most belong to the alpha- and alphaA-conotoxin families.) When administered to mice and fish in vivo, alphaC-PrXA caused paralysis and death. In electrophysiological assays, alphaC-PrXA potently antagonized mouse muscle nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.8 and 3.0 nM for the adult (alpha1beta1epsilondelta subunits) and fetal (alpha1beta1gammadelta subunits) muscle nAChR subtypes, respectively. When tested on a variety of ligand-gated and voltage-gated ion channels, alphaC-PrXA proved to be a highly specific inhibitor of the neuromuscular nAChR. The peptide competes with alpha-bungarotoxin for binding at the alpha/delta and alpha/gamma subunit interfaces of the nAChR, with higher affinity for the alpha/delta subunit interface. alphaC-PrXA is strikingly different from the many conopeptides shown to be nicotinic antagonists; it is most similar in its general biochemical features to the snake toxins known as Waglerins.

    27 June, 2007

      Conantokin peptide action at NMDA receptors

      Sheng Z, Dai Q, Prorok M, Castellino FJ. (2007) Subtype-selective antagonism of N-methyl-d-aspartate receptor ion channels by synthetic conantokin peptides. Neuropharmacology. 53: 145-156.
      W.M. Keck Center for Transgene Research, 230 Raclin-Carmichael Hall, University of Notre Dame, Notre Dame, IN 46556, USA; Department of Chemistry and Biochemistry, 230 Raclin-Carmichael Hall, University of Notre Dame, Notre Dame, IN 46556, USA.

      Abstract: Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.

    25 June, 2007

      Characterizing disulfide-rich peptides

      Quinton L, Demeure K, Dobson R, Gilles N, Gabelica V, Pauw ED. (2007) New Method for Characterizing Highly Disulfide-Bridged Peptides in Complex Mixtures: Application to Toxin Identification from Crude Venoms. J Proteome Res. 6: 3216-3223.
      Laboratoire de Spectrométrie de Masse, Centre d'Analyse des Résidus en Traces, Université de Liège, Liège B-4000, Belgium, and CEA/DSV/iBiTec-S/SIMOPRO, Gif-sur-Yvette Cedex 91191, France.

      Abstract:Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series. Keywords: In-source decay * post-source decay * conotoxin * snake toxins * disulfide-bridged peptides * top-down sequencing.

      Gammahydroxyvaline in Conus gladiator and Conus mus peptides

      Cudic M, Mari F, Fields GB. (2007) Synthesis and Solid-Phase Application of Suitably Protected gamma-Hydroxyvaline Building Blocks. J Org Chem. 72:5581-5586
      Department of Chemistry & Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431.

      Abstract: Recently, an unexpected modified residue, gamma-hydroxy-d-valine (d-Hyv), was identified within ribosomally expressed polypeptide chains of four conopeptides from the venoms of Conus gladiator and Conus mus. To assemble Hyv-containing peptides, we have explored several routes for the synthesis of appropriately functionalized Hyv building blocks. d-Hyv was produced from d-Val by using a variation of the previously published K2PtCl4/CuCl2 oxidative method. Direct synthesis of Boc- or Cbz-d-Hyv lactone proceeded in low yield; additionally, the lactones are too unreative for solid-phase applications. 9-Borabicyclononane or copper-complexed d-Hyv was prepared and treated with tert-butyldimethylsilyl trifluoromethanesulfonate (TBDMSOTf) to produce d-Hyv(O-TBDMS). The most efficient complex disruption was achieved by Chelex 110 resin (Na+ form) treatment of copper-complexed d-Hyv(O-TBDMS). Reaction of d-Hyv(O-TBDMS) with Fmoc-OSu produced Fmoc-d-Hyv(O-TBDMS) in 26% yield from d-Val. The Fmoc-d-Hyv(O-TBDMS) diastereomers were separated by preparative RP-HPLC in 13% yield from d-Val. Fmoc-d-Hyv(O-TBDMS) was used for the synthesis of the conopeptide gld-V* from Conus gladiator. The isolated synthetic and natural products had coincidental mass and NMR spectra. The methodology presented herein will greatly facilitate biological studies of Hyv-containing sequences, such as receptor responses to hydroxylated versus nonhydroxylated conopeptides and the relative susceptibility of proteins to modification by oxidative stress.

    16 June, 2007

      Novel conopeptide NMB-1 blocks pressure-evoked pain

      Drew LJ, Rugiero F, Cesare P, Gale JE, Abrahamsen B, Bowden S, Heinzmann S, Robinson M, Brust A, Colless B, Lewis RJ, Wood JN. (2007) High-threshold mechanosensitive ion channels blocked by a novel conopeptide mediate pressure-evoked pain. PLoS ONE. 2007 Jun 13;2:e515.
      Drew LJ, Rugiero F, Cesare P, Gale JE, Abrahamsen B, Bowden S, Heinzmann S, Robinson M, Brust A, Colless B, Lewis RJ, Wood JN.
      Department of Biology, University College London, London, United Kingdom.

      Abstract: Little is known about the molecular basis of somatosensory mechanotransduction in mammals. We screened a library of peptide toxins for effects on mechanically activated currents in cultured dorsal root ganglion neurons. One conopeptide analogue, termed NMB-1 for noxious mechanosensation blocker 1, selectively inhibits (IC(50) 1 microM) sustained mechanically activated currents in a subset of sensory neurons. Biotinylated NMB-1 retains activity and binds selectively to peripherin-positive nociceptive sensory neurons. The selectivity of NMB-1 was confirmed by the fact that it has no inhibitory effects on voltage-gated sodium and calcium channels, or ligand-gated channels such as acid-sensing ion channels or TRPA1 channels. Conversely, the tarantula toxin, GsMTx-4, which inhibits stretch-activated ion channels, had no effects on mechanically activated currents in sensory neurons. In behavioral assays, NMB-1 inhibits responses only to high intensity, painful mechanical stimulation and has no effects on low intensity mechanical stimulation or thermosensation. Unexpectedly, NMB-1 was found to also be an inhibitor of rapid FM1-43 loading (a measure of mechanotransduction) in cochlear hair cells. These data demonstrate that pharmacologically distinct channels respond to distinct types of mechanical stimuli and suggest that mechanically activated sustained currents underlie noxious mechanosensation. NMB-1 thus provides a novel diagnostic tool for the molecular definition of channels involved in hearing and pressure-evoked pain.

      Sequence comparison:
      FNWRCCLIPCRRNHKKFFC conopeptide analogue NMB-1
      FNWRCCLIPACRRNHKKF_C conotoxin rho-TIA from Conus tulipa

      Conotoxins from Conus achatinus

      Liu L, Chew G, Hawrot E, Chi C, Wang C. (2007) Two potent alpha3/5 conotoxins from piscivorous Conus achatinus. Acta Biochim Biophys Sin (Shanghai). 39:438-444.
      Institute of Protein Research, Tongji University, Shanghai 200092, China. chunguangwang@mail.tongji.edu.cn

      Abstract: Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. alpha3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of alpha3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac1.1a and Ac1.1b, with appropriate disulfide bonding, based on cDNA sequences ofalpha3/5conotoxins from C. achatinus. Ac1.1a and Ac1.1b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC50 values of 36 nM and 26 nM, respectively. For Ac1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the N-terminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the alpha1-delta subunit interface instead of the alpha1-gamma binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.

    11 June, 2007

      Novel toxin-like peptides

      Kaplan N, Morpurgo N, Linial M. (2007) Novel Families of Toxin-like Peptides in Insects and Mammals: A Computational Approach. J Mol Biol. 369: 553-566.
      Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.

      Abstract: Most animal toxins are short proteins that appear in venom and vary in sequence, structure and function. A common characteristic of many such toxins is their apparent structural stability. Sporadic instances of endogenous toxin-like proteins that function in non-venom context have been reported. We have utilized machine learning methodology, based on sequence-derived features and guided by the notion of structural stability, in order to conduct a large-scale search for toxin and toxin-like proteins. Application of the method to insect and mammalian sequences revealed novel families of toxin-like proteins. One of these proteins shows significant similarity to ion channel inhibitors that are expressed in cone snail and assassin bug venom, and is surprisingly expressed in the bee brain. A toxicity assay in which the protein was injected to fish induced a strong yet reversible paralytic effect. We suggest that the protein may function as an endogenous modulator of voltage-gated Ca(2+) channels. Additionally, we have identified a novel mammalian cluster of toxin-like proteins that are expressed in the testis. We suggest that these proteins might be involved in regulation of nicotinic acetylcholine receptors that affect the acrosome reaction and sperm motility. Finally, we highlight a possible evolutionary link between venom toxins and antibacterial proteins. We expect our methodology to enhance the discovery of additional novel protein families.

    6 June, 2007

      Conotoxins from Conus virgo

      Mandal AK, Ramasamy MR, Sabareesh V, Openshaw ME, Krishnan KS, Balaram P. (2007) Sequencing of T-Superfamily Conotoxins from Conus virgo: Pyroglutamic Acid Identification and Disulfide Arrangement by MALDI Mass Spectrometry. J Am Soc Mass Spectrom 18:1396-404
      Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

      Abstract: De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH(2), and Vi1361, ZCCPTMPECCRI-NH(2), which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of w(n)- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

    29 May, 2007

      Ziconotide: a review

      McGivern, JG (2007) Ziconotide: a review of its pharmacology and use in the treatment of pain. Neuropsychiatric Disease and Treatment. 3: 69-85.
      HTS-Molecular Pharmacology, Amgen Inc., Thousand Oaks, CA, USA. Correspondence: Joseph G McGivern HTS-Molecular Pharmacology, Amgen Inc., Thousand Oaks, CA 91320, USA Email: mcgivern@amgen.com

      Abstract: Ziconotide is a powerful analgesic drug that has a unique mechanism of action involving potent and selective block of N-type calcium channels, which control neurotransmission at many synapses. The analgesic efficacy of ziconotide likely results from its ability to interrupt pain signaling at the level of the spinal cord. Ziconotide is a peptidic drug and has been approved for the treatment of severe chronic pain in patients only when administered by the intrathecal route. Importantly, prolonged administration of ziconotide does not lead to the development of addiction or tolerance. The current review discusses the various studies that have addressed the in vitro biochemical and electrophysiological actions of ziconotide as well as the numerous pre-clinical studies that were conducted to elucidate its antinociceptive mechanism of action in animals. In addition, this review considers the pivotal Phase 3 (and other) clinical trials that were conducted in support of ziconotide's approval for the treatment of severe chronic pain and tries to offer some insights regarding the future discovery and development of newer analgesic drugs that would act by a similar mechanism to ziconotide but which might offer improved safety, tolerability and ease of use.

    24 May, 2007

      Isolation of alpha-Conotoxin MII-sensitive nicotinic receptor subtypes (alpha6* )

      Salminen O, Drapeau J, McIntosh M, Collins AC, Marks MJ, Grady SR. (2007) Pharmacology of {alpha}-Conotoxin MII-sensititive subtypes of nicotinic acetylcholine receptors isolated by breeding of null mutant mice. . Mol Pharmacol. 71: 1563-1571.
      Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado (O.S., J.A.D., E.L., M.C., A.C.C., M.J.M., S.R.G.); and Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.)

      Abstract: Subtypes of nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits comprise 25-30% of the presynaptic nAChRs expressed in striatal dopaminergic terminals in rodents and 70% in monkeys. This class of receptors, potentially important in nicotine addiction, binds alpha-conotoxin MII (alpha-CtxMII) with high affinity, and is heterogeneous, consisting of several subtypes in mice, possibly an important consideration for the design of compounds that selectively activate or antagonize the alpha6 subclass of nAChRs. Selected null mutant mice were bred to generate isolated subtypes of alpha6beta2*nAChRs expressed in vivo for assessing pharmacology of alpha6beta2* nAChRs. Binding to striatal membranes and function in synaptosomes from (alpha4-/-)(beta3+/+) and (alpha4-/-)(beta3-/-) mice were measured and compared to wildtype (alpha4+/+)(beta3+/+) mice. Gene deletions (alpha4 and beta3) decreased binding of [(125)I]-alpha-CtxMII without affecting affinity for alpha-CtxMII, or inhibition of alpha-CtxMII binding by epibatidine or nicotine. Deletion of the alpha4 subunit substantially increased EC50 values for both nicotine and cytisine stimulated alpha-CtxMII-sensitive dopamine release from striatal synaptosomes. A further increase in EC50 values was seen upon the additional deletion of the beta3 subunit. The data indicate that one alpha-CtxMII-sensitive nAChR subtype, prevalent on wildtype dopaminergic terminals, has the lowest EC50 for a nicotine-mediated function so far measured in mice. In conclusion, the gene deletion strategy enabled isolation of alpha6* subtypes, and these nAChR subtypes exhibited differential activation by nicotine and cytisine.

    23 May, 2007

      Contulakin-G (CGX-1160): Pharmacokinetics

      Kern SE, Allen J, Wagstaff J, Shafer SL, Yaksh T. (2007) The pharmacokinetics of the conopeptide Contulakin-G (CGX-1160) after intrathecal administration: An analysis of data from studies in beagles. Anesth Analg. 104:1514-120.
      Department of Pharmaceutics, University of Utah, 421 Wakara Way #318, Salt Lake City, Utah 84108. Steven.Kern@hsc.utah.ed.

      Abstract: BACKGROUND: The synthetic peptide agent Contulakin-G (CGX-1160), isolated from the toxin of the snail Conus geographus, produces significant analgesia in animals. Its peptide structure requires intrathecal administration for effectiveness, therefore we determined the intrathecal pharmacokinetics of CGX-1160 after bolus dose and multiple day infusions to beagles. METHODS: For the bolus dose study, eight animals received a dose ranging from 16.7 to 1000 nmol under isoflurane anesthesia. Cerebral spinal fluid sampling for drug assay occurred up to 24 h. For the multiple day infusion study, three animals received infusions of 10, 40, and 160 mug/h respectively for 24 h at each rate. Cerebral spinal fluid sampling occurred during the infusion rate and the washout period after the 72 h of cumulative drug delivery. Data from the two study designs were modeled separately using NONMEM. RESULTS: The results showed a biexponential disposition profile for both experiments with a rapid rate constant that was an order of magnitude greater than the slow rate constant. The bolus results showed a nonlinear dependence of the slow rate constant on administered dose due to the large bolus range used in the study. CONCLUSION: These data, coupled with clinical pharmacology results, provide a basis for determining appropriate dosing strategies to achieve therapeutic intrathecal concentrations of Contulakin-G.

      Contulakin-G (CGX-1160): Antinociceptive activity in rats and dogs

      Allen JW, Hofer K, McCumber D, Wagstaff JD, Layer RT, McCabe RT, Yaksh TL. (2007) An assessment of the antinociceptive efficacy of intrathecal and epidural contulakin-G in rats and dogs. Anesth Analg. 104:1505-1513.
      Anesthesiology Research, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0818. tyaksh@ucsd.ed.

      Abstract: Contulakin-G is a novel conopeptide with an incompletely defined mechanism of action. To assess nociceptive activity we delivered Contulakin-G as a bolus intrathecally (0.03, 0.1, 0.3, 3 nmol) or epidurally (10, 30, 89 nmol) in rats. Intrathecal Contulakin G significantly decreased Phase II and, to a lesser degree, Phase I paw flinching produced by intradermal formalin. Intrathecal and epidural doses of ED50s were 0.07 nmol and 45 nmol, respectively, giving an epidural/intrathecal ED(50) ratio = 647). In dogs, intrathecal Contulakin-G (50-500 nmoL) produced a dose-dependent increase in the thermally evoked skin twitch latency by 30 min after administration, as did morphine (150 and 450 nmol). Epidural morphine (750 and 7500 nmol), but not epidural 1000 nmol Contulakin-G, also significantly decreased skin twitch in dogs. No changes in motor function were seen in any rats or dogs receiving these doses of Contulakin-G. In dogs, no physiologically significant dose-dependent changes in motor function, heart rate, arterial blood pressure, or body temperature were found. Contulakin-G is a potent antinociceptive drug when delivered intrathecally with no observable negative side effects in rats or dogs and may provide an alternative to opioid spinal analgesics.

    17 May, 2007

      Conantokin antagonism of NMDA receptor

      Prorok M, Castellino FJ. (2007) The Molecular Basis of Conantokin Antagonism of NMDA Receptor Function. Curr Drug Targets. 8: 633-642.
      Department of Chemistry and Biochemistry and the W. M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, Indiana 46556, USA. mprorok@nd.edu

      Abstract: The N-methyl-D-aspartate receptor (NMDAR), a subtype of ionotropic glutamate receptor, has been implicated in a host of chronic and acute neurological disorders. Accordingly, much emphasis has been placed on the development of safe and effective therapeutic agents that specifically antagonize this target. The conantokins are a class of small, naturally occurring peptides that inhibit ion flow through the NMDAR. Some conantokins demonstrate receptor subunit selectivity, a pharmacological attribute of emerging importance in the search for suitable drug candidates. The current review summarizes the NMDAR inhibitory properties of the conantokins, including structure-function relationships and mechanism of action. This information is fundamental to the rational design of suitable agents that can effectively treat pathophysiologies linked to NMDAR dysfunction.

    16 May, 2007

      alpha-CtxArIB[V11A,V16D], from Conus arenatus, is a highly selective alpha7 nicotinic receptor ligand

      Whiteaker P, Christensen S, Yoshikami D, Dowell C, Watkins M, Gulyas J, Rivier J, Olivera BM, McIntosh JM. (2007) Discovery, Synthesis, and Structure Activity of a Highly Selective alpha7 Nicotinic Acetylcholine Receptor Antagonist. Biochemistry. 46: 6628-6638.
      Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah 84112, and Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037.

      Abstract: Nicotinic acetylcholine receptors (nAChRs) that contain an alpha7 subunit are widely distributed in neuronal and nonneuronal tissue. These receptors are implicated in the release of neurotransmitters such as glutamate and in functions ranging from thought processing to inflammation. Currently available ligands for alpha7 nAChRs have substantial affinity for one or more other nAChR subtypes, including those with an alpha1, alpha3, alpha6, and/or alpha9 subunit. An alpha-conotoxin gene was cloned from Conus arenatus. Predicted peptides were synthesized and found to potently block alpha3-, alpha6-, and alpha7-containing nAChRs. Structure-activity information regarding conotoxins from distantly related Conus species was employed to modify the C. arenatus derived toxin into a novel, highly selective alpha7 nAChR antagonist. This ligand, alpha-CtxArIB[V11A,V16D], has low nanomolar affinity for rat alpha7 homomers expressed in Xenopus laevis oocytes, and antagonism is slowly reversible. Kinetic analysis provided insight into the mechanism of antagonism. alpha-CtxArIB interacts with five ligand binding sites per alpha7 receptor, and occupation of a single site is sufficient to block function. The peptide was also shown to be highly selective in competition binding assays in rat brain membranes. alpha-CtxArIB[V11A,V16D] is the most selective ligand yet reported for alpha7 nAChRs.

    28 April, 2007

      Peptide-polymer hybrids of Conotoxin SIIIA enhance its analgesic properties

      Green BR, Catlin P, Zhang MM, Fiedler B, Bayudan W, Morrison A, Norton RS, Smith BJ, Yoshikami D, Olivera BM, Bulaj G. (2007) Conotoxins containing nonnatural backbone spacers: cladistic-based design, chemical synthesis, and improved analgesic activity. Chem Biol. 14: (4), 399-407.
      Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

      Abstract: Disulfide-rich neurotoxins from venomous animals continue to provide compounds with therapeutic potential. Minimizing neurotoxins often results in removal of disulfide bridges or critical amino acids. To address this drug-design challenge, we explored the concept of disulfide-rich scaffolds consisting of isostere polymers and peptidic pharmacophores. Flexible spacers, such as amino-3-oxapentanoic or 6-aminohexanoic acids, were used to replace conformationally constrained parts of a three-disulfide-bridged conotoxin, SIIIA. The peptide-polymer hybrids, polytides, were designed based on cladistic identification of nonconserved loci in related peptides. After oxidative folding, the polytides appeared to be better inhibitors of sodium currents in dorsal root ganglia and sciatic nerves in mice. Moreover, the polytides appeared to be significantly more potent and longer-lasting analgesics in the inflammatory pain model in mice, when compared to SIIIA. The resulting polytides provide a promising strategy for transforming disulfide-rich peptides into therapeutics.

      See also:
      Neuropathic Pain Treatment Gives Avigen New Lease on Life and Neuromed Follows the Snail Trail Chemistry & Biology, Volume 14, Issue 4, 20 April 2007, Pages 341-343 by Wendy Wolfson.

      Sodium channels: Role in neuropathic pain. Review

      Hargus NJ, Patel MK. (2007) Voltage-gated Na+ channels in neuropathic pain. Expert Opin Investig Drugs. 2007 May;16(5):635-646.
      University of Virginia Health System, Department of Anesthesiology, Neuroscience Graduate Program, 1 Hospital Drive, Old Medical School, Charlottesville, VA 22908, USA.

      Abstract: Neuropathic pain occurs as a result of some form of injury to the nervous system. Although the basis of the disease remains to be fully elucidated, numerous studies have suggested a major role for ion channels in the pathogenesis of neuropathic pain. As Na+ channels play a fundamental role in not only the generation but also in the conduction of an action potential, they have received considerable attention in the aetiology of pain sensation and have become important pharmacological targets. In this review, the authors discuss the importance of specific Na+ channel isoforms in the pathophysiology of neuropathic pain and the present use of Na+ channel antagonists in the treatment of neuropathic pain.

    23 April, 2007

      Alpha-conotoxin BuIA from Conus bullatus

      Jin AH, Brandstaetter H, Nevin ST, Tan CC, Clark RJ, Adams DJ, Alewood PF, Craik DJ, Daly NL. (2007) Structure of alpha-conotoxin BuIA: Influences of disulfide connectivity on structural dynamics. BMC Struct Biol.7: 28

      Abstract: BACKGROUND: Alpha-Conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in alpha-conotoxins is variable, leading to the classification of sub-families. BuIA is the only alpha-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin. RESULTS: In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer. CONCLUSION: These findings are opposite to the general trends observed for alpha-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.

    19 April, 2007

      M-2 and M-4 conotoxins adopt 'flying bird' backbone conformation.

      Du WH, Han YH, Huang FJ, Li J, Chi CW, Fang WH. (2007) Solution structure of an M-1 conotoxin with a novel disulfide linkage. FEBS J. 274: 2596-2602
      Departments of Chemistry, Renmin University of China and Beijing Normal University, Beijing, China.

      Abstract: The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C-CC-), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M-superfamily family can be divided into four branches, namely M-1, -2, -3 and -4. Recently, two M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M-2 and M-4 branches. Here we report the 3D structure of mr3e determined by 2D (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six varphi dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 +/- 0.19 A. Although mr3e has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a 'flying bird'. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold.

    15 April, 2007

      Chi-conotoxin MrIA from Conus marmoreus binds at the mouth of the norepinephrine transporter

      Paczkowski FA, Sharpe IA, Dutertre S, Lewis RJ. (2007) chi -Conopeptide and tricyclic antidepressant interactions at the norepinephrine transporter define a new transporter model. J Biol Chem. 282: 17837-17844
      Institute for Molecular Bioscience, Brisbane, Queensland 4072.

      Abstract: Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide chi-MrIA is a noncompetitive and highly selective inhibitor of the NE transporter (NET) and is being developed as a novel intrathecal analgesic. We used site-directed mutagenesis to generate a suite of mutated transporters to identify two amino acids (Leu469 and Glu382) that affected the affinity of chi-MrIA to inhibit [3H]NE uptake through human NET. Residues that increased the Kd of a tricyclic antidepressant (nisoxetine) were also identified (F207, S225, H296, T381 and D473). F207, S225, H296 and T381 also affected the rate of NE transport without affecting NE Km. In a new model of NET constructed from the bLeuT crystal structure, chi-MrIA-interacting residues were located at the mouth of the transporter near residues affecting the binding of small molecule inhibitors.

      Erythromelalgia - a painful peripheral disorder may be due to a defect in Nav1.7 sodium channels in sensory neurons.

      Sheets PL, Jackson Ii JO, Waxman SG, Dib-Hajj S, Cummins TR.(2007) A Nav1.7 Channel Mutation Associated with Hereditary Erythromelalgia Contributes to Neuronal Hyperexcitability and Displays Reduced Lidocaine Sensitivity. J Physiol. 581(Pt 3): 1019-1031
      Indiana University School of Medicine.

      Abstract: Mutations in the TTX-sensitive voltage-gated sodium channel subtype Nav1.7 have been implicated in the painful inherited neuropathy, hereditary erythromelalgia. Hereditary erythromelalgia can be difficult to treat and, although sodium channels are targeted by local anesthetics such as lidocaine, some patients do not respond to treatment with local anesthetics. This study examined electrophysiological differences in Nav1.7 caused by a hereditary erythromelalgia mutation (N395K) that lies within the local anesthetic binding site of the channel. The N395K mutation produced a hyperpolarized voltage-dependence of activation, slower kinetics of deactivation, and impaired steady-state slow inactivation. Computer simulations indicate that the shift in activation is the major determinant of the hyperexcitability induced by erythromelalgia mutations in sensory neurons, but that changes in slow inactivation can modulate the overall impact on excitability. This study also investigated lidocaine inhibition of the Nav1.7-N395K channel. We show that the N395K mutation attenuates the inhibitory effects of lidocaine on both resting and inactivated Nav1.7. The IC50 for lidocaine was estimated at 500 microM for inactivated wild-type Nav1.7 and 2.8 mM for inactivated Nav1.7-N395K. The N395K mutation also significantly reduced use-dependent inhibition of lidocaine on Nav1.7 current. In contrast, a different hereditary erythromelalgia mutation (F216S), not located in the local anesthetic binding site, had no effect on lidocaine inhibition of Nav1.7 current. Our observation of reduced lidocaine inhibition on Nav1.7-N395K shows that the residue N395 is critical for lidocaine binding to Nav1.7 and suggests that the response of individuals with hereditary erythromelalgia to lidocaine treatment may be determined, at least in part, by their specific genotype.

      - see also -

      Dobbs, D. (2007) The Pain Gate. Scientific American Mind 18: (2), 48-55.

      Abstract and Extract: The article presents information about the physiological mechanism of erythromelalgia. Patients suffering from this disorder experience searing heat in the feet and lower legs and sometimes in the hands. In erythromelalgia, a defect in a sodium channel in pain-sensing neurons in the legs and arms makes the neurons overexcitable. The neurons overreact and sends signals of blazing pain even in the absence of tissue damage.

      "....For most of the 140 years since it was named, the disorder known as burning man syndrome has operated in near-total obscurity. Even today it afflicts perhaps 200 to 500 people in all of North America and a few thousand worldwide. Until about three years ago, essentially all medical knowledge about it was contained in its name, erythromelalgia, which translates as "painful red extremities." Few doctors knew of it, only a handful had seen it, and none knew what caused it or how to treat it. At any given time, the few thousand people who had it suffered its torment--searing heat in the feet and lower legs and sometimes in the hands--without understanding why. Most thought they were completely alone.
      Pam Costa, 42, lived her first decade this way. She is one of perhaps 30 or 40 people in the U.S., and possibly 200 to 500 worldwide, known to have an inherited form of the disease. .. "

      Futher reading:

    • Mutations in SCN9A, Encoding a Sodium Channel Alpha Subunit, in Patients with Primary Erythermalgia. Y. Yang, Y. Wang, S. Li, Z. Xu, H. Li, L. Ma, J. Fan, D. Bu, B. Liu, Z. Fan, G. Wu, J. Jin, B. Ding, X. Zhu and Y. Shen in Journal of Medical Genetics, Vol. 41, No. 3, pages 171-174; March 2004.

    • The Erythromelalgia Association provides more research information at: http://www.erythromelalgia.org/

    5 April, 2007

      alpha-CtxMII E11A identifies alpha6alpha4beta2*beta3* nicotinic receptor population as selectively vulnerable in Parkinsons

      Bordia T, Grady SR, McIntosh JM, Quik M. (2007) Nigrostriatal damage preferentially decreases a subpopulation of {alpha}6{beta}2* nAChRs in mouse, monkey and Parkinson's disease striatum. Mol Pharmacol. 72: 52-61.
      The Parkinson's Institute.

      Abstract: Parkinson's disease is a neurodegenerative movement disorder characterized by a loss of substantia nigra dopamine neurons, and corresponding declines in molecular components present on striatal dopaminergic nerve terminals. These include the alpha6beta2* nicotinic receptors (nAChRs), which are localized exclusively on dopamine terminals in striatum (*denotes the presence of possible additional subunits). Here, we used a novel alpha-conotoxin MII (alpha-CtxMII) analog E11A to further investigate alpha6beta2* nAChR subtypes in mouse, monkey and human striatum. Receptor competition studies with (125)I-alpha-CtxMII showed that E11A inhibition curves were biphasic, suggesting the presence of two distinct alpha6beta2* nAChR subtypes. These include a very high (fM) and a high (pM) affinity site, with ~40% of the sites in the very high affinity form. Interestingly, only the high affinity form was detected in alpha4 nAChR null mutant mice. Since (125)I-alpha-CtxMII binds primarily to alpha6alpha4beta2*beta3* and alpha6beta2*beta3* nAChR subtypes in mouse striatum, these data suggest that the population lost in the alpha4 knockout mice was the alpha6beta2*beta3* subtype. We next investigated the effect of nigrostriatal lesioning on these two striatal alpha6beta2*populations in two animal models and in Parkinson's disease. There was a preferential loss of the very high affinity subtype in striatum of MPTP-treated mice, MPTP-treated monkeys and Parkinson's disease cases. These data suggest that dopaminergic terminals expressing the alpha6alpha4beta2*beta3* population are selectively vulnerable to nigrostriatal damage. This latter nAChR subtype, identified with alpha-CtxMII E11A, may therefore provide a unique marker for dopaminergic terminals particularly sensitive to nigrostriatal degeneration in Parkinson's disease.

    4 April, 2007

      Evolution of alpha-conotoxins

      Yuan DD, Han YH, Wang CG, Chi CW. (2007) From the identification of gene organization of alpha conotoxins to the cloning of novel toxins. Toxicon. 49: 1135-1149
      Institute of Protein Research, TongJi University, 1239 Siping Road, Shanghai 200092, China.

      Abstract: In the venoms of cone snails, alpha conotoxins are competitive antagonists of nicotinic acetylcholine receptors. Eleven novel cDNA and eight partial gene sequences (including two pseudogenes) of alpha conotoxins were identified from five species of cone snail. As expected, every cDNA encodes a precursor of prepropeptide. In all the partial genes of alpha conotoxins identified, there is a long intron inserted at a fixed position in the pro-region, dividing the encoding region into two exons. The mutation rate in exon I (encoding the signal peptide and a part of pro-region) is much lower than that in exon II (encoding the other part of pro-region, the mature peptide and 3' untranslational region). Interestingly, the sequences at the 5' and 3' end of introns are highly conserved. In addition, in the identified introns exist long dinucleotide (e.g. "GT", "CA") or trinucleotide ("CAT") repeats. In the special case of Pu1.1, there are five almost identical repeats of a 150bp sequence in the long intron. Taking advantage of the conserved 3' end sequence of intron, 16 alpha conotoxins, as well as a pseudogene and three kappaA conotoxins, were identified from their genomic DNAs. Based on the comparison of these cDNA and gene sequences, a hypothesis of the alpha conotoxin evolution was proposed.

      Conus textile yields novel T-superfamily conotoxins

      Luo S, Zhangsun D, Zhang B, Chen X, Feng J. (2006) Direct cDNA cloning of novel conotoxins of the T-superfamily from Conus textile. Peptides. 27: 2640-2646.
      Key Laboratory for Tropical Biological Resources (MOE), Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou Hainan 570228, China. luosulan2003@163.com

      Abstract: The T-superfamily is a large and diverse group of peptides, widely distributed in venom ducts of all major feeding types of Conus. These peptides are likely to be functionally diverse. A directed PCR-based approach using primers based on the conserved signal sequence was applied to investigate new conotoxins of the T-superfamily from Conus textile native to Hainan. Using RT-PCR and 3'-RACE, four novel cDNA sequences encoding precursor peptides were identified in C. textile. They share a common T-superfamily cysteine pattern (CC-CC, with two disulfide bridges). The predicted peptides are small (9-12 amino acids). TeAr193 composed of nine amino acid residues is one of the shortest T-superfamily conotoxins ever found. Patterns of sequence divergence and Cys codon usage define the major T-superfamily branches and suggest how these separate branches arose. The sequences of the signal regions exhibited highest conservation, whereas the sequences of the mature peptides were either almost identical or highly divergent; and conservation of the pro-region was intermediate between that observed in signal and toxin regions. The elucidated cDNAs of the four toxins will facilitate a better understanding of the relationship between structure and function.

      Conopeptides from Indian Conidae with therapeutic promise

      Gowd KH, Sabareesh V, Sudarslal S, Iengar P, Franklin B, Fernando A, Dewan K, Ramaswami M, Sarma SP, Sikdar S, Balaram P, Krishnan KS. (2005) Novel peptides of therapeutic promise from Indian Conidae. Ann N Y Acad Sci. 1056: 462-734. Review
      Tata Institute of Fundamental Research, Colaba, Mumbai, 40005, India.

      Abstract: Highly structured small peptides are the major toxic constituents of the venom of cone snails, a family of widely distributed predatory marine molluscs. These animals use the venom for rapid prey immobilization. The peptide components in the venom target a wide variety of membrane-bound ion channels and receptors. Many have been found to be highly selective for a diverse range of mammalian ion channels and receptors associated with pain-signaling pathways. Their small size, structural stability, and target specificity make them attractive pharmacologic agents. A select number of laboratories mainly from the United States, Europe, Australia, Israel, and China have been engaged in intense drug discovery programs based on peptides from a few snail species. Coastal India has an estimated 20-30% of the known cone species; however, few serious studies have been reported so far. We have begun a comprehensive program for the identification and characterization of peptides from cone snails found in Indian Coastal waters. This presentation reviews our progress over the last 2 years. As expected from the evolutionary history of these venom components, our search has yielded novel peptides of therapeutic promise from the new species that we have studied.

    31 March, 2007

      Synthesis of dicarba analogues of Conotoxin ImI

      Robinson AJ, Elaridi J, Van Lierop BJ, Mujcinovic S, Jackson WR. (2007) Microwave-assisted RCM for the synthesis of carbocyclic peptides.J Pept Sci. 13: 280-285
      School of Chemistry, Monash University, Clayton 3800, Victoria, Australia.

      Abstract:Microwave irradiation dramatically improves the efficiency of ring closing metathesis (RCM) reactions of resin-attached peptides and the technology is illustrated by the highly selective synthesis of dicarba analogues of alpha-conotoxin ImI. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

    28 March, 2007

      O-superfamily conotoxins targeting voltage-gated sodium channels

      Heinemann SH, Leipold E. (2007) Conotoxins of the O-superfamily affecting voltage-gated sodium channels. Cell Mol Life Sci. 64(11):1329-1340
      Center of Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena, Drackendorfer Str. 1, 07747, Jena, Germany, Stefan.H.Heinemann@uni-jena.de.

      Abstract: The venoms of predatory cone snails harbor a rich repertoire of peptide toxins that are valuable research tools, but recently have also proven to be useful drugs. Among the conotoxins with several disulfide bridges, the O-superfamily toxins are characterized by a conserved cysteine knot pattern: C-C-CC-C-C. While omega-conotoxins and kappa-conotoxins block Ca(2+) and K(+) channels, respectively, the closely related delta- and muO-conotoxins affect voltage-gated Na(+) channels (Na(v) channels). delta-conotoxins mainly remove the fast inactivation of Na(v) channels and, thus, functionally resemble long-chain scorpion alpha-toxins. muO-conotoxins are functionally similar to mu-conotoxins, since they inhibit the ion flowthrough Na(v) channels. Recent results from functional and structural assays have gained insight into the underlying molecular mechanisms. Both types of toxins are voltage-sensor toxins interfering with the voltage-sensor elements of Na(v) channels.

      Conotoxin alpha-PIB from Conus purpurascens

      Lopez-Vera E, Jacobsen RB, Ellison M, Olivera BM, Teichert RW. (2007) A novel alpha conotoxin (alpha-PIB) isolated from C. purpurascens is selective for skeletal muscle nicotinic acetylcholine receptors. Toxicon. 49: 1193-1199;
      Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

      Abstract: The alpha-conotoxin family is comprised of peptides that share the following arrangement of cysteine residues in the primary amino acid sequence: -CC-C-C-, where each dash represents a variable number of amino acids. The number of amino acids between cysteine residues has been used to group the alpha-conotoxins into distinct subfamilies. These subfamilies include the alpha4/7-, alpha4/3- and alpha3/5-conotoxins, so named for the number of amino acids between 2nd/3rd and 3rd/4th cysteine residues, respectively. The alpha3/5-conotoxins antagonize vertebrate-muscle nicotinic acetylcholine receptors (nAChRs), while the alpha4/7- and alpha4/3-conotoxins primarily inhibit vertebrate neuronal nAChRs. To date, these three subfamilies are the most extensively characterized of the alpha-conotoxin family. Here we report the purification and characterization of an unusual alpha4/4-conotoxin, alpha-conotoxin PIB (alpha-PIB), from the venom of Conus purpurascens, with the following amino-acid sequence: ZSOGCCWNPACNKNRC (Z=pyroglutamate, O=hydroxyproline). This peptide demonstrates high affinity inhibition of vertebrate-muscle nAChRs, and paralytic effects when injected in vivo. Testing of alpha-PIB against other receptors indicated that the inhibitory effect is specific for skeletal muscle nAChRs. alpha-PIB shares the key biochemical and pharmacological characteristics of the alpha-conotoxin family.

    13 March, 2007

      Conantokin interactions

      Dai Q, Sheng Z, Geiger JH, Castellino FJ, Prorok M. (2007) Helix-helix interactions between homo- and heterodimeric gamma-carboxyglutamate-containing conantokin peptides and their derivatives. J Biol Chem.282: 12641-12649
      Department of Chemistry and Biochemistry and The W.M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN 46556.

      Abstract: The conantokins are a family of small, naturally-occuring, gamma-carboxyglutamate (Gla)-rich peptides that specifically antagonize the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor. One member of this family, conantokin-G (con-G), undergoes Ca2+-mediated self-assembly to form an antiparallel helical dimer. Subunit interactions in this complex are incumbent upon intermolecular Ca(2+)-bridging of Gla residues spaced at i, i+4, i +7, i +11 intervals within the monomer. Herein, we further probe the molecular determinants governing such helix-helix interactions. Select variants were synthesized to evaluate the contributions of non-Gla residues to conantokin self-association. Con-G dimerization was shown to be exothermic and accompanied by positive heat capacity changes. Using positional Gla variants of conantokin-R (con-R), a non-dimerizing conantokin, i, i+4, i +7, i +11 Gla spacing alone was shown to be insufficient for self-assembly. The Ca(2+)-dependent antiparallel heterodimerization of con-G and con-T(K7gamma), two peptides that harbor optimal Gla spacing, was established. Lastly, the effects of covalently-constrained con-G dipeptides on NMDA-evoked current in HEK293 cells expressing combinations of NR1a, NR1b, NR2A, and NR2B subunits of the NMDA receptor were investigated. The antiparallel dipeptide was unique in its ability to potentiate current at NR1a/2A receptors and, like monomeric con-G, was inhibitory at NR1a/2B and NR1b/2B combinations. In contrast, the parallel species was completely inactive at all subunit combinations tested. These results suggest that, under physiological Ca(2+) concentrations, equilibrium levels of con-G dimer most likely exist in an antiparallel orientation and exert effects on NMDA receptor activity that differ from the monomer.

      Conantokin stabilization via a "metallo-zipper"

      Cnudde SE, Prorok M, Dai Q, Castellino FJ, Geiger JH. (2007) The crystal structures of the calcium-bound con-G and con-T[K7gamma] dimeric peptides demonstrate a metal-dependent helix-forming motif. J Am Chem Soc. 129:1586-93.
      Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.

      Abstract: Short peptides that have the ability to form stable alpha-helices in solution are rare, and a number of strategies have been used to produce them, including the use of metal chelation to stabilize folding of the backbone. However, no example exists of a structurally well-defined helix stabilized exclusively through metal ion chelation. Conantokins (con)-G and -T are short peptides that are potent antagonists of N-methyl-D-aspartate receptor channels. While con-G exhibits no helicity alone, it undergoes a structural transition to a helical conformation in the presence of a variety of multivalent cations, especially Mg2+ and Ca2+. This complexation also results in antiparallel dimerization of two peptide helices in the presence of Ca2+, but not Mg2+. A con-T variant, con-T[K7gamma], displays very similar behavior. We have solved the crystal structures of both Ca2+/con-G and Ca2+/con-T [K7gamma] at atomic resolution. These structures clearly show the nature of the metal-dependent dimerization and helix formation and surprisingly also show that the con-G dimer interface is completely different from the con-T[K7gamma] interface, even though the metal chelation is similar in the two peptides. This represents a new paradigm in helix stabilization completely independent of the hydrophobic effect, which we define as the "metallo-zipper."

    5 March, 2007

      Conopressin peptide in Conus villepinii venom

      Moller C, Mari F. (2007)A vasopressin/oxytocin related conopeptide with a Gla at position 8. Biochem J. 404: 413-419
      Department of Chemistry & Biochemistry, Centre of Excellence in Biomedical and Marine Biotechnology, Florida Atlantic University, 777 Glades Rd., Boca Raton, 33431 Florida, USA.

      Abstract: Vasopressins and oxytocins are homologous, ubiquitous and multifunctional peptides present in animals. Conopressins are vasopressin/oxytocin related peptides that have been found in the venom of cone snails, a genus of marine predatory mollusks that envenom their prey with a complex mixture of neuroactive peptides. Here, we report the purification and characterization of a unique conopressin isolated from the venom of Conus villepinii, a vermivorous cone snail species from the Western Atlantic Ocean. This novel peptide, designated gamma-conopressin-vil, has the sequence: CLIQDCPgammaG* (gamma = Gla). The unique feature of this vasopressin/oxytocin-like peptide is that the eighth residue is a gamma-carboxyglutamate instead of a neutral or basic residue; therefore, it could not be directly classified into either the vasopressin or the oxytocin peptide families. NanoNMR spectroscopy of the peptide directly isolated from the cone snails revealed that the native gamma-conopressin-vil undergoes structural changes in the presence of calcium. This suggests that the peptide binds calcium, and the calcium-binding process is mediated by the Gla residue. However, the negatively charged residues in the sequence of gamma-conopressin-vil may mediate calcium-binding by a novel mechanism not observed in other peptides of this family.

    25 February, 2007

      125I-Labelled alpha-conotoxin MII used to localize alpha6beta2* neuronal nicotinic receptors

      Khwaja M, McCormack A, McIntosh JM, Di Monte DA, Quik M.(2007) Nicotine partially protects against paraquat-induced nigrostriatal damage in mice; link to alpha6beta2* nAChRs. J Neurochem. 100: 180-190.
      The Parkinson's Institute, Sunnyvale, CA 94089, USA.

      Abstract: Epidemiological studies indicate that smoking is a negative, and exposure to pesticides, a positive risk factor for Parkinson's disease (PD). The purpose of this study was to assess the interplay between these two factors in a rodent model of nigrostriatal damage. To approach this, mice were administered nicotine, the agent in smoke implicated in neuroprotection. They were then treated for 3 weeks with the pesticide, paraquat, while nicotine was continued. Paraquat treatment decreased (25%) nigral dopaminergic neurons, consistent with previous results. Chronic nicotine administration significantly protected against nigral cell damage, with only a 16% decline in mice treated with both nicotine and paraquat. Paraquat treatment also decreased (14%) the striatal dopamine transporter, an effect that was partially prevented by nicotine. These changes in the striatal dopamine transporter paralleled those in a select striatal alpha6beta2* nicotinic receptor (nAChR) subtype. In contrast, striatal alpha4beta2* nAChRs were not decreased with paraquat treatment, suggesting they are on a differential subset of dopaminergic terminals. The results show that nicotine treatment partially protects against paraquat-induced declines in nigrostriatal dopaminergic neurons to which a select population of alpha6beta2* nAChRs are localized. Moreover, these data support epidemiological findings that environmental influences can elicit opposing effects on nigrostriatal dopaminergic integrity.

      Determinants of protein folding in conotoxins

      Kang TS, Radic Z, Talley TT, Jois SD, Taylor P, Kini RM. (2007) Protein Folding Determinants: Structural Features Determining Alternative Disulfide Pairing in alpha- and chi/lambda-Conotoxins. Biochemistry 46: 3338-3355
      Protein Science Laboratory, Department of Biological Sciences, and Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117 543, Department of Pharmacology, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, and Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 13298.

      Abstract: alpha-Conotoxins isolated from Conus venoms contain 11-19 residues and preferentially fold into the globular conformation that possesses a specific disulfide pairing pattern (C1-3, C2-4). We and others isolated a new family of chi-conotoxins (also called lambda conotoxins) with the conserved cysteine framework of alpha-conotoxins but with alternative disulfide pairing (C1-4, C2-3) resulting in the ribbon conformation. In both families, disulfide pairing and hence folding are important for their biological potency. By comparing the structural differences, we identified potential structural determinants responsible for the folding tendencies of these conotoxins. We examined the role of conserved proline in the first intercysteine loop and the conserved C-terminal amide on folding patterns of synthetic analogues of ImI conotoxin by comparing the isoforms with the regiospecifically synthesized conformers. Deamidation at the C-terminus and substitution of proline in the first intercysteine loop switch the folding pattern from the globular form of alpha-conotoxins to the ribbon form of chi/lambda-conotoxins. The findings are corroborated by reciprocal folding of CMrVIA chi/lambda-conotoxins. Substitution of Lys-6 from the first intercysteine loop of CMrVIA conotoxin with proline, as well as the inclusion of an amidated C-terminal shifted the folding preference of CMrVIA conotoxin from its native ribbon conformation toward the globular conformation. Binding assays of ImI conotoxin analogues with Aplysia and Bulinus acetylcholine binding protein indicate that both these substitutions and their consequent conformational change substantially impact the binding affinity of ImI conotoxin. These results strongly indicate that the first intercysteine loop proline and C-terminal amidation act as conformational switches in alpha- and chi/lambda-conotoxins.

    23 February, 2007

      Ziconotide in clinical use - a Review

      Lynch SS, Cheng CM, Yee JL. (2006) Intrathecal ziconotide for refractory chronic pain. Ann Pharmacother. 40: 1293-1300. Review.
      Department of Clinical Pharmacy, University of California, San Francisco, 94143, USA. lynchs@pharmacy.ucsf.edu

      Abstract: OBJECTIVE: To describe the pharmacology, efficacy, and safety of ziconotide for treatment of severe chronic pain in patients who are candidates for intrathecal therapy. DATA SOURCES: A PubMed/MEDLINE search (1966-June 2006) was conducted using the terms ziconotide, Prialt, and SNX-111. Manufacturer-provided data, the Food and Drug Administration medical review of ziconotide, and abstracts presented at American Pain Society meetings (2001-2006) were also reviewed. STUDY SELECTION AND DATA EXTRACTION: Human studies evaluating the efficacy and safety of ziconotide for the treatment of chronic pain were considered. Animal data were excluded. DATA SYNTHESIS: Ziconotide is the first and only neuronal-type (N-type) calcium-channel blocker. Ziconotide must be administered intrathecally via continuous infusion. A programmable implanted variable-rate microinfusion device, or an external microinfusion device and catheter must be utilized. In double-blind, placebo-controlled studies, ziconotide significantly improved patient perception of pain from baseline to the end of the study periods, which ranged from 11 to 21 days. Patients enrolled in clinical trials were intolerant of or refractory to other treatment modalities. There have been no studies that directly compared ziconotide with other intrathecal or systemic analgesics. Key ziconotide-related adverse events are neuropsychiatric, including depression, cognitive impairment, and hallucinations; depressed levels of consciousness; and elevation of creatine kinase levels. Ziconotide is also associated with a risk of meningitis due to possible contamination of the microinfusion device. CONCLUSIONS: Ziconotide is a therapeutic option for treatment of severe chronic pain in patients who have exhausted all other agents, including intrathecal morphine, and for whom the potential benefit outweighs the risks of serious neuropsychiatric adverse effects and of having an implanted device. Further studies are needed to determine the comparative efficacy of ziconotide and other pain therapies.

    17 February, 2007

      omega-conotoxins CVID and MVIIA: Use in a rat model of inflammatory pain

      Rycroft, B.K., Vikman, K.V. and Christie, M.J. (2007) Inflammation reduces the contribution of N-type calcium channels to primary afferent synaptic transmission onto NK1 receptor positive lamina I neurons in the rat dorsal horn. J. Physiol. 580(Pt.3): 883-894
      The University of Sydney, Sydney, Australia
      * To whom correspondence should be addressed. E-mail: macc@med.usyd.edu.au.

      Abstract: N-type calcium channels contribute to release of glutamate from primary afferent terminals synapsing onto nocisponsive neurons in the dorsal horn of the spinal cord but little is known of functional adaptations to these channels in persistent pain states. Subtype-selective conotoxins and other drugs were used to determine the role of different calcium channel types in a rat model of inflammatory pain. Electrically-evoked primary afferent synapses onto lumber dorsal horn neurons were examined three days after induction of inflammation with intraplantar Complete Freund's Adjuvant. The maximal inhibitory effect of the N-type calcium channel blockers, omega-conotoxins CVID and MVIIA, were attenuated in NK1 receptor positive lamina I neurons after inflammation but the potency of CVID was unchanged. This was associated with reduced inhibition of the frequency of asynchronous-evoked synaptic events by CVID studied in the presence of extracellular strontium, suggesting reduced N-type channel contribution to primary afferent synapses after inflammation. After application of CVID, the relative contributions of P/Q and L channels to primary afferent transmission and the residual current were unchanged by inflammation suggesting the adaptation was specific to N-type channels. Blocking T-type channels did not affect synaptic amplitude under control or inflamed conditions. Reduction of N-type channel contribution to primary afferent transmission was selective for NK1-receptor positive neurons identified by post-hoc immunohistochemistry and did not occur at synapses in laminae IIo or IIi, or inhibitory synapses. These results suggest that inflammation selectively down-regulates N-type channels in the terminals of primary afferents synapsing onto (presumed) nociceptive lamina I NK1-receptor positive neurons.
      Key words: Calcium (Ca2+) channels • Dorsal horn • Pain

      New postage stamps depicting cone shells

      During 2006, 20 new stamps depicting 18 species of Conus were released. Twelve of these species had not previously been depicted on stamps (C. hieroglyphus (Dominica), and from the Solomon Islands, C.auratinus, C.consors, C.sucatus f. magdalenae, C.sulcatus f. brettinghami, C.ochroleucus f. tmetus, C.aureus, C.corallinus, C.consors f.poehlianus, C.proximus and C.canonicus. Tom Walker again kindly provided me with the images. They have been added to the Cone Shells on Stamps resource and are available for viewing by clickng here.

    14 February, 2007

      alpha-conotoxin MI interactions with nicotinic receptor.

      Cortez L, Marino-Buslje C, de Jimenez Bonino MB, Hellman U. (2007) Interactions between alpha-conotoxin MI and the Torpedo marmorata receptor alpha-delta interface. Biochem Biophys Res Commun. 355: 275-279.
      Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires Instituto de Quimica y Fisicoquimica Biologicas (UBA, CONICET) Junin 956 (1113) Buenos Aires, Argentina.

      Abstract: The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jimenez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.

    2 February, 2007

      Ziconotide treatment of CRPS TypeI.

      Stanton-Hicks M, Kapural L. (2006) An effective treatment of severe complex regional pain syndrome type 1 in a child using high doses of intrathecal ziconotide. J Pain Symptom Manage. 32: 509-511. No abstract available.
      Michael Stanton-Hicks, MD and Leonardo Kapural, MD, PhD are from the Pain Management Department, The Cleveland Clinic Foundation, Cleveland, Ohio, USA.

      Extract: "This observation suggests that ziconotide may be an effective therapy for advanced CRPS type 1 and that it can be used in the pediatric patient population. It appears that slow titration of ziconotide may still bring benefits in suppression of allodynia and pain relief, even at doses of 24 mg/day, without producing obvious side effects".

      High throughput synthesis of conotoxins.

      Brust A, Tickle AE. (2007) High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format. J Pept Sci. 13: 133-141.
      Xenome Ltd, 120 Meiers Road, Indooroopilly 4068, Australia.

      Abstract: Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides. Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of >95% purity. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

      Predicting conotoxin superfamilies.

      Lin H, Li QZ. (2007) Predicting conotoxin superfamily and family by using pseudo amino acid composition and modified Mahalanobis discriminant. Biochem Biophys Res Commun. 354: 548-551
      Laboratory of Theoretical Biophysics, Department of Physics, College of Sciences and Technology, Inner Mongolia University, Hohhot 010021, PR China.

      Abstract: The conotoxin proteins are disulfide rich small peptides that target ion channels and G protein coupled receptors. And they provide promising application in treating some chronic pain, epilepsy, cardiovascular diseases, and so on. Conotoxins may be classified into 11 superfamilies: A, D, I1, I2, J, L, M, O, P, S, and T according to the disulfide connectivity, highly conserved N-terminal precursor sequence and similar mode of actions. Successful prediction mature conotoxin superfamily peptide has important signification for the biological and pharmacological functions of the toxins. In this study, a new algorithm of increment of diversity combined with modified Mahalanobis discriminant is presented to predict five superfamilies by using the pseudo amino acid composition. The results of jackknife cross-validation test show that the overall prediction sensitivity and specificity are 88% and 91%, respectively. The predictive algorithm is also used to predict three O-conotoxin families. The 72% sensitivity and 78% specificity are obtained. These results indicate that the conotoxin superfamily peptides correlate with their amino acid compositions.

    27 January, 2007

      Ziconotide (omega-conotoxin MVIIA)- short review

      Klotz U. (2006) Ziconotide--a novel neuron-specific calcium channel blocker for the intrathecal treatment of severe chronic pain--a short review. Int J Clin Pharmacol Ther. 44: 478-483. Review.
      Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany. ulrich.klotz@ikp-stuttgart.de

      Abstract: Worldwide a large number of patients suffer from severe chronic pain even after treatment with opioids following the 3-step analgesic ladder developed by the WHO. Intraspinal agents, including morphine, have been tried as a fourth step. However, approximately 20% of cases remain refractory. Ziconotide, an intrathecal analgesic with orphan drug status, is a novel alternative for the management of chronic intractable pain. Ziconotide is a synthetic peptide based on the toxin of the fish-hunting marine snail, Conus magus. It is the first therapeutic agent in a new pharmacological class of "topically" active analgesics that selectively target neuron-specific (N-type), voltage-gated calcium channels. Ziconotide produces potent analgesia by interruption of Ca-dependent primary afferent transmission of pain signals in the spinal cord. Ziconotide was significantly more effective than placebo in the treatment of chronic malignant (p < 0.001) and non-malignant pain (p < 0.001). In several clinical studies morphine dosages could be substituted by ziconotide. The drug has a lag-time for the onset and offset of analgesia and adverse effects. Initial doses should therefore be low (2.4 microg/day) and titrated slowly (increasing up to a maximum of 21.6 microg/day in increases of 2.4 microg/day no more than twice weekly). The gradual increase in dose helps to reduce the incidence and severity of adverse events which affect primarily the central nervous system (e.g. dizziness, nausea, confusion). Ziconotide maintains its analgesic efficacy over months and does not cause tolerance, dependence or respiratory depression. Following intrathecal infusion ziconotide is distributed within the cerebral spinal fluid (CSF) where its clearance (0.38 ml/min) corresponds to the rate of turnover of the CSF. Negligible amounts of ziconotide are present in the systemic circulation where it is rapidly degraded by proteolysis. In conclusion, ziconotide is a new and valuable alternative analgesic for the acute and long-term treatment of severe pain, especially in patients refractory to opioids.

    23 January, 2007

      Cone shells on Solomon Is. stamps

      The Solomon Islands have released a set of beautiful cone stamps - look at http://www.casb.co.uk/news/news.asp?step=4&NewsID=881 if you'd like to see the whole set.
      Values: 5c Conus marmoreus; 10c Conus auratinus; 20c Conus ferrugineus ; 50c Conus consors; 80c Conus magdalenae; 90c Conus sulcatus brettinghami; $1 Conus tmetus; $1.50 Conus aureus; $2 Conus corallinus; $3 Conus floccatus; $4 Conus panniculus; $10 Conus pohlianus; $20 Conus proximus; $50 Conus canonicus. Release Date: 31st October 2006

    19 January, 2007

      Conus envenmomation

      Haddad V 2nd, de Paula Neto JB, Cobo VJ. (2006) Venomous mollusks: the risks of human accidents by Conus snails (gastropoda: conidae) in Brazil. Rev Soc Bras Med Trop. 39:498-500.
      Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP. haddadjr@fmb.unesp.br

      Abstract: Mollusks of the genus Conus present a venomous apparatus composed of radulae, a chitin structure linked to glands, which injects potent neurotoxic peptides, causing serious human envenomation and even death, associated with the blockage of certain receptors and muscular paralysis. No reported envenomation has occurred in Brazil, but certain populations are at risk of accidents. To download full article (.pdf) click here
      Rev Soc Bras Med Trop. 39:498-500

    15 January, 2007

      Contryphans from C.loroisii and C. amadis

      Sabareesh V, Gowd KH, Ramasamy P, Sudarslal S, Krishnan KS, Sikdar SK and Balaram P. (2007) Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels. Peptides. 27:2647-2654.
      Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

      Abstract: Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis,a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O:4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.

      omega-Conotoxin GVIA inhibits sperm motility

      Li L, Liu J, Li J, Ye Z. (2006) Pharmacological investigation of voltage-dependent Ca2+ channels in human ejaculatory sperm in vitro. J Huazhong Univ Sci Technolog Med Sci. 26: 607-609.
      Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

      Abstract: The types of the voltage-dependent calcium channels (VDCCs) in human ejaculatory sperm and the effects of calcium channel blocker (CCB) on human sperm motility parameters in vitro were investigated. The human sperm motility parameters in vitro in response to the pharmacological agents nifedipine (NIF, inhibitor of L-type VDCC) and omega-conotoxin (GVIA, inhibitor of N-type VDCC) were compared and analyzed statistically. The results showed that NIF (1, 5, 10 micromol/L) could not only significantly affect human sperm's shape but also spermatozoa motility after incubated at least 10 min in vitro (P<0.001). GVIA (0.1, 0.5 and 1 micromol/L) could just only significantly affect human sperm's progressive motility (a %+b %) after incubated for 20 min in vitro (P<0.01), but they both could not significantly affect spermic abnormality rate. It is suggested that L-type VDCC, non L-type VDCCs and isoform of L-type VDCC exist in the cell membrane of human sperm solely or together, and they participate in the spermic physiological processes especially the spermic motility.

    10 January, 2007

      Conotoxin MVIIA (Ziconotide) is a potential therapeutic agent for temporal lobe epilepsy

      Wang S, Ding M, Wu D, Zhan J, Chen Z. (2007) omega-Conotoxin MVIIA inhibits amygdaloid kindled seizures in Sprague-Dawley rats. Neurosci Lett. 413: 163-167.
      Departments of Pharmacology and Biochemistry, School of Medicine, Zhejiang University, Hangzhou 310058, China; Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.

      Abstract: omega-Conotoxin MVIIA (omega-CTX MVIIA) is a reversible and potent antagonist of N-type voltage-dependent calcium channels (VDCCs) in neurons. In this study, we evaluated the effect of a fusion form of omega-CTX MVIIA with glutathione S-transferase (GST) on amygdaloid kindled seizures. Intracerebraventricular (i.c.v.) injection of the fusion protein of GST-omega-CTX MVIIA significantly decreased seizure stage and shortened afterdischarge duration and generalized seizure duration in a dose-dependent and time-related manner. In addition, GST-omega-CTX MVIIA significantly increased the GABA levels in the cortex and glycine levels in the brainstem. In contrast, GST alone did not have any effect on seizure behavior or neurochemical levels. These findings firstly demonstrate that the N-type VDCC is a potential therapeutic target for temporal lobe epilepsy. The mechanism of the anticonvulsant function of omega-CTX MVIIA is related to the blockade of N-type VDCC-mediated neurotransmitter release in the brain.

      Pain relief after transplantation of human neuronal cells

      Eaton MJ, Wolfe SQ, Martinez M, Hernandez M, Furst C, Huang J, Frydel BR, Gomez-Marin O. (2007) Subarachnoid Transplant of a Human Neuronal Cell Line Attenuates Chronic Allodynia and Hyperalgesia After Excitotoxic Spinal Cord Injury in the Rat. J Pain. 2007 Jan;8(1):33-50.

      VA RR&D Center of Excellence in Functional Recovery in Chronic Spinal Cord Injury, VAMC, Miami, Florida.; The Miami Project to Cure Paralysis, Miller School of Medicine, University of Miami, Miami, Florida.; Department of Neurosurgery, Miller School of Medicine, University of Miami, Miami, Florida. Abstract: The relief of neuropathic pain after spinal cord injury (SCI) remains daunting, because pharmacologic intervention works incompletely and is accompanied by multiple side effects. Transplantation of human cells that make specific biologic agents that can potentially modulate the sensory responses that are painful would be very useful to treat problems such as pain. To address this need for clinically useful human cells, the human neuronal NT2 cell line was used as a source to isolate a unique human neuronal cell line that synthesizes and secretes/releases the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine. This new cell line, hNT2.17, expresses an exclusively neuronal phenotype, does not incorporate bromodeoxyuridine during differentiation, and does not express the tumor-related proteins fibroblast growth factor 4 and transforming growth factor-alpha during differentiation after 2 weeks of treatment with retinoic acid and mitotic inhibitors. The transplant of predifferentiated hNT2.17 cells was used in the excitotoxic SCI pain model, after intraspinal injection of the mixed AMPA/metabotropic receptor agonist quisqualic acid (QUIS). When hNT2.17 cells were transplanted into the lumbar subarachnoid space, tactile allodynia and thermal hyperalgesia induced by the injury were quickly and potently reversed. Control cell transplants of nonviable hNT2.17 cells had no effect on the hypersensitivity induced by QUIS. The effects of hNT2.17 cell grafts appeared 1 week after transplants and did not diminish during the 8-week course of the experiment when grafts were placed 2 weeks after SCI. Immunohistochemistry and quantification of the human grafts were used to ensure that many grafted cells were still present and synthesizing GABA at the end of the study. These data suggest that the human neuronal hNT2.17 cells can be used as a "biologic minipump" for antinociception in models of SCI and neuropathic pain. PERSPECTIVE: This study describes the initial characterization and use of a human-derived cell line to treat neuropathic pain that would be suitable for clinical application, once further tested for safety and approved by the Food and Drug Administration. A dose of these human cells could be delivered with a spinal tap and affect the intrathecal spinal environment for sensory system modulation.


    5 January, 2007

    Welcome to 2007 - and good shelling everyone !!


    Conus victoriae, Broome, Western Australia. Collected by Sally Johnsen and Bruce Livett, on 7 October 2006. Photographed by David Paul, in marine aquarium, Dept. Zoology, University of Melbourne. Note the egg sacks. Click on each image for a larger version.


      New Conus Newsletter

      The Cone Collector is a new electronic newsletter of general interest to all with an interest in cone shells. The first issue 'The Cone Collector #0',(Editor, Antonio Monteiro) and published in October 2006, oddly begins at #0 and was warmly received by a selected group of cone shell enthusiasts. This was quickly followed in January 2007 by 'The Cone Collector #1'. The founding Editorial Committee comprised Antonio Monteiro from Portugal and Paul H. Kersten from the Netherlands. A list of topics will whet your appetite:

        In 'The Cone Collector #0', pp. 3-4, Antonio Monteiro has written an Obituary describing some notable contributions of the late Antonio Jose (Bob) da Motta. Preempting questions about the role of 'The Cone Collector', there follows an FAQ. Then a very interesting article by A.M. "About the Nomenclature of Angolan Cones". Then follows "Cone Reign Supreme" and a Cone Bibliography reviewing the major books on cones. This includes a review of the recently published (2005) book on Cones (In Russian - KOHYCbl) or "Cones - deadly mollusks of tropical seas. Catalogue of the cone shells collection of the State Darwin Museum, Moscow." A most useful "List of Species Described after 1999" pp. 15-16, compiled by Paul Kersten, is follwoed by "The Work of Hwass" with a Plate illustrating four cones and completes this issue of 17 pages.

        In 'The Cone Collector #1', 28 pages !, topics included 'Who Created Conidae", "Who's Who in Cones" (showcasing William J. Fenzan), an article on "Modern Classification" and a "Letter from Mike Filmer" recollecting about first meeting Bob da Motta. There follows "Photos of live C. curassaviensis Hwass, 1792, which is brilliantly illustrated, after which is a comment from John Tucker "About the Nomenclature of Angolan Cones". There is a description of "A population of Conus ventricosus Gmelin, 1791 south of Taranto, Ionian Sea" by Giancarlo Paganelli, complete with bibliography and photos. "An Artist's Impressions" presents artist Boet van Heugten with two renditions of Conus peli, Moolenbeek, 1996. Marco Bettocchi contributes an article on "The Genus Problem" followed by a contribution "The Empire Strikes Back" by Alfred J. Spoo. The cone book Bibliography is continued followed by "What Am I?" where comment is sought on some unusual specimens. Photos of Special Specimens are requested. Jason Biggs contributed an article on "Conus gloriamaris discovered in Vanuatu" with photos of one very angry specimen extending its proboscis. John Tucker from Great Rivers Field Station, Illinois Natural History Survey, has provided "A bibliography of cone shells described after 1999". The list of recent cones has been expanded in 'The Cone Collector #1', January 2007, to include alphabetic listings of "Species described from 1995 to 2000" pp. 20-23 and "Species described from 2000 to 20006" pp. 23-28. These listings are illustrated with photos of selected cones. Contributions to future issues and comments on the articles published to date are welcomed by the Editor.

        Editorial Rules are provided in 'The Cone Collector #0'. To request to be added to the e-list to receive "The Cone Collector" send and e-mail to the Editor, Antonio Monteiro or write snail-mail to him at Rua Carlos Calisto, 3-4o Esq., 1400-043 Lisboa, Portugal, or even phone ([00-351]-965115923) during daylight hours !


      Ziconotide - a Review

      Wallace MS. (2006) Ziconotide: a new nonopioid intrathecal analgesic for the treatment of chronic pain. Expert Rev Neurother. 6:1423-1428. Review.
      Professor of Clinical Anesthesiology, Program Director, Center for Pain Medicine, University of California, San Diego, 9500 Gilman Drive, 0924, San Diego, CA 92093, USA. mswallace@ucsd.edu

      Abstract: Ziconotide is a new nonopioid intrathecal agent recently approved for the treatment of chronic pain. Ziconotide is indicated for the management of severe chronic pain in patients for whom intrathecal therapy is warranted and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies or intrathecal morphine. Ziconotide blocks the N-type calcium channels located in the superficial dorsal horn of the spinal cord, resulting in potent analgesia. The efficacy of ziconotide has been demonstrated in three randomized, placebo-controlled trials in over 500 patients. In addition, its safety has been demonstrated in over 1200 subjects. Ziconotide is a potent analgesic with a narrow therapeutic window. The drug requires a slow titration in order to achieve analgesia while avoiding dose-limiting side effects. This review examines the currently available information on this new analgesic.

      Pain, Calcium channels, Prialt - and more

      Cao YQ. (2006) Voltage-gated calcium channels and pain. Pain. 126: 5-9. Review.
      Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, MO 63110, United States. caoy@morpheus.wustl.edu

      Introduction: Virtually all excitable cells express plasma membrane voltage-gated Ca2+ channels (VGCCs) that transduce electrical activity into intracellular biochemical signals. Membrane depolarization triggers the opening of VGCCs to allow rapid influx of extracellular Ca2+, which, in turn, regulates numerous physiological processes: the release of neurotransmitters and neuropeptides, neuronal excitability and plasticity, gene expression, development as well as cell survival and death (Tsien and Wheeler, 1999). Studies of Ca2+ currents in dorsal root ganglion (DRG) neurons date back to the early days of VGCC research and in 1985 led to the functional discovery of N-type Ca2+ channel (Nowycky et al., 1985). Remarkably, only twenty years later, the N-type Ca2+ channel blocker Prialt, also known as SNX111 or ziconotide, was approved in both United States and Europe to treat intractable pain. It is anticipated that additional treatment modalities will derive from further understanding of the contribution of multiple VGCCs to nociceptive processing, in both the normal and the disease setting.




    Continued in What's new in 2006
    See also : What's new in 2005, What's new in 2004, What's new in 2003, What's new in 2002, What's new in 2001, What's new in 2000, What's new in 1999, What's new in 1998, What's New in 1997 and What's New in 1996

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    Continued in What's new in 2006

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